WO2016197339A1

WO2016197339A1 - Method for preparing polyelectrolyte capsule and prepared polyelectrolyte capsule

Info

Publication number: WO2016197339A1
Application number: PCT/CN2015/081135
Authority: WO
Inventors: 何丽贞; 蔡轩昂
Original assignee: 聚和国际股份有限公司
Priority date: 2015-06-10
Filing date: 2015-06-10
Publication date: 2016-12-15

Abstract

Disclosed are a method for preparing a polyelectrolyte capsule and a prepared polyelectrolyte capsule. The method comprises the following steps: (A) obtaining a porous particle containing a porous material; and (B) mixing the porous particle with a polysaccharide and a peptide to obtain a core-shell particle; wherein the core of the core-shell particle is in the porous particle, and the shell of the core-shell particle contains the polysaccharide and the peptide.

Description

Preparation method of polyelectrolyte capsule and polyelectrolyte capsule obtained thereby

Technical field

The invention relates to a preparation method of a polyelectrolyte capsule and a polyelectrolyte capsule obtained thereby, in particular to a polyelectrolyte capsule prepared from two polyelectrolyte polymers.

Background technique

Polyelectrolyte refers to a polymer whose repeat unit contains an electrolyte group which can be dissociated in an aqueous solution to charge the polymer. Polyelectrolytes have excellent water solubility and thus have many biochemical and medical applications, for example, as a component of a drug carrier to form a shell of a core-shell particle drug (this drug carrier can be called a polyelectrolyte capsule, which has a higher Water soluble). Existing polyelectrolyte housings are typically multi-layered structures formed by alternating positively charged polyelectrolytes and negatively charged polyelectrolytes. The preparation process of such a polyelectrolyte capsule is rather cumbersome, and the positively charged polyelectrolyte and the negatively charged polyelectrolyte are required to interact with the core in order to form a thin layer on the periphery of the core by electrostatic attraction. After each reaction between the polyelectrolyte and the core, it needs to be washed and separated to carry out the next reaction of the polyelectrolyte and the core, so the process is rather cumbersome and time consuming. In addition, since a plurality of processes of washing and centrifugation are required in the process, it is difficult to avoid the loss of active components in the polyelectrolyte capsule, making it difficult for the polyelectrolyte capsule to satisfy the demand for medical application.

In order to solve the problem that the polyelectrolyte capsule is too cumbersome in the preparation process and the active component is lost, it has been attempted in the art to simultaneously add a positively charged polyelectrolyte and a negatively charged polyelectrolyte to interact with the core to form an outer membrane. However, such an attempt has not been successful because the positively charged polyelectrolyte and the negatively charged polyelectrolyte easily attract each other in the solution to accumulate and precipitate, and it is impossible to form a thin film on the periphery of the core efficiently. Accordingly, there is a need in the art for a novel capsule of an outer membrane formed of a polyelectrolyte for use in the medical field.

Summary of the invention

It is an object of the present invention to provide a polyelectrolyte capsule which provides excellent water solubility and is thus used as a pharmaceutical carrier.

Another object of the present invention is to provide a method for preparing a polyelectrolyte capsule, which is simpler to prepare than the prior polyelectrolyte capsule, and saves time and cost.

In order to achieve the foregoing object, the present invention provides a method for preparing a polyelectrolyte capsule comprising the following steps:

(A) obtaining porous particles comprising a porous material;

(B) mixing the particles with a polysaccharide and a peptide to obtain core-shell particles; wherein the core of the core-shell particles is the porous And a shell comprising the polysaccharide and the peptide; wherein the polysaccharide and the peptide have a weight average molecular weight of not more than 5,000 Daltons.

Preferably, the polysaccharide and the peptide have a weight average molecular weight of between 1,000 and 3,500 Daltons.

Preferably, the polysaccharide and the peptide are linked to each other by a covalent bond.

Preferably, the polysaccharide has a dissociation constant pK _{b of} from 7.5 to 12.

Preferably, the sugar is: chitin, trimethyl chitosan, cationic starch, or a combination thereof.

Preferably, the peptide has a dissociation constant pKa of from 3 to 5.

Preferably, the peptide is: polyglutamic acid, polyaspartic acid, or a combination thereof.

Preferably, the porous material comprises: calcium carbonate, calcium superphosphate (Ca(H ₂ PO ₄ ) ₂ ), silicon dioxide, manganese carbonate, cadmium carbonate, polystyrene, melamine formaldehyde, polylactic acid polyglycol Acid (PLGA), polylactic acid (PLA), or a combination thereof.

Preferably, before the step (A), the porous active material is further filled with the first active ingredient.

Preferably, the step (B) further comprises the step (C): (C) dissolving the core.

Preferably, the polyelectrolyte capsule comprises an interior space defined by the shell, wherein the method of preparation further comprises filling the interior space with a first active ingredient.

Preferably, the first active ingredient comprises: doxorubicin, folic acid, bovine serum albumin, insulin, or a combination thereof.

Preferably, before the step (B), the second active ingredient is further grafted onto the polysaccharide and/or the peptide.

Preferably, the second active ingredient comprises: a dye, a metal, an antibody, a receptor, or a combination thereof.

The present invention further provides a polyelectrolyte capsule comprising: a shell; and an inner space defined by the shell; wherein the shell comprises a glycopeptide; wherein the glycopeptide comprises: a polysaccharide and a peptide; The polysaccharide and the peptide are linked to each other by a bond; wherein the glycopeptide has a weight average molecular weight of not more than 10,000 Daltons.

Preferably, the glycopeptide has a weight average molecular weight of from 2,000 to 7,000 Daltons.

Preferably, the polysaccharide is: chitin, trimethyl chitosan, cationic starch, or a combination thereof.

Preferably, the peptide dissociation constant pK _a of 3 to 5.

Preferably, the interior space comprises a first active ingredient.

Preferably, the inner space comprises a core comprising porous particles and interconnected with the shell by electrostatic attraction; wherein the porous particles comprise a porous material.

Preferably, the porous material is filled with a first active ingredient.

Preferably, the shell is conjugated with a second active ingredient; wherein the second active ingredient comprises: a dye, a metal, an antibody, a receptor, or a combination thereof.

Preferably, the bond is: a covalent bond, a hydrogen bond, or an ionic bond. Preferably, the covalent bond is an amide bond.

In summary, the present invention provides a novel polyelectrolyte capsule preparation method and a polyelectrolyte capsule obtained by the method. The method of the invention has simple steps, can save time and cost, and can avoid the loss of active ingredients in the cumbersome process.

DRAWINGS

Fig. 1 shows a scanning electron micrograph of calcium carbonate particles having a radius of 1 μm prepared in Example 1.

2 shows a scanning electron micrograph of calcium carbonate particles having a radius of 5 μm prepared in Example 1.

Fig. 3 shows Fourier transform infrared spectroscopy of doxromycin-filled calcium carbonate particles, unfilled calcium carbonate particles, and doxorubicin in Example 1.

Figure 4 shows the embedding rate of doxorubicin in Example 1 at 1 hour and 16 hours.

Fig. 5 shows a Fourier transform infrared spectrum of calcium carbonate particles filled with folic acid, unfilled calcium carbonate particles, and folic acid in Example 1.

Fig. 6 shows a Fourier transform infrared spectrum of calcium carbonate particles, BSA, and unfilled calcium carbonate particles filled with BSA in Example 1.

Fig. 7 shows a Fourier transform infrared spectrum, (A) calcium carbonate particles filled with insulin and unfilled calcium carbonate particles in Example 1, and (B) calcium carbonate particles filled with insulin and insulin in Example 1.

Fig. 8 is a photomicrograph showing a copolymerization (yoke) of a polyelectrolyte capsule (glycopeptide and calcium carbonate particles not filled with an active ingredient) obtained in Experiment D1 of Example 1. (A) calcium carbonate particles having a radius of 1 μm; (B) calcium carbonate particles having a radius of 5 μm.

Figure 9 shows a confocal micrograph of a polyelectrolyte capsule (glycopeptide and doxorubicin-loaded calcium carbonate particles) prepared in Experiment D1 of Example 1.

Figure 10 shows a confocal micrograph of a polyelectrolyte capsule (glycopeptide and folic acid-filled calcium carbonate particles) prepared in Experiment D1 of Example 1.

Figure 11 shows a confocal micrograph of a polyelectrolyte capsule (glycopeptide and calcium carbonate albumin-filled calcium carbonate particles) prepared in Experiment D1 of Example 1. (A) calcium carbonate particles having a radius of 1 μm; (B) calcium carbonate particles having a radius of 5 μm.

Figure 12 shows a confocal micrograph of a polyelectrolyte capsule (glycopeptide and insulin-filled calcium carbonate particles) prepared in Experiment D1 of Example 1.

Figure 13 shows a scanning electron micrograph of the solution of the calcium carbonate nucleus in Experiment D2 of Example 1. (A) before dissolution; (B) after dissolution.

Figure 14 shows the doxorubicin release rate in Experiment E of Example 2.

detailed description

The present invention relates to a polyelectrolyte capsule. By virtue of the fact that the polyelectrolyte is charged in an aqueous solution, the polyelectrolyte capsule of the present invention can be used as a pharmaceutical carrier to enhance the water solubility of the active ingredient, thereby improving the efficacy of the active ingredient.

The "active ingredient" as used in the present invention means a component having a desired activity. For example, if the goal is to treat cancer, the active ingredient refers broadly to all substances that have cancer cell suppression (eg, Doxorubicin). By way of further example, if the objective is to treat diabetes, the active ingredient generally refers to all substances (eg, insulin) that are capable of preventing, alleviating, and/or treating diabetes. By way of further example, if the target is a specific molecule in the organism under the microscope field of view, the active ingredient may be a fluorescent protein. In other words, the active ingredient of the present invention broadly refers to a target-oriented ingredient having the desired activity.

In view of the disadvantages of the prior art polyelectrolyte capsules, such as time consuming, cumbersome operation steps, and low filling degree of active ingredients, an aspect of the present invention provides a method for preparing a polyelectrolyte capsule, which comprises the following steps: (A) obtaining a porous a particle comprising a porous material; (B) mixing the particle with a polysaccharide (first polyelectrolyte) and a peptide (second polyelectrolyte) to obtain core-shell particles; wherein the core of the core-shell particle is Porous particles, and the shell of the core-shell particles comprises the polysaccharide and the peptide. In a possible embodiment, the porous material comprises: calcium carbonate, calcium superphosphate (Ca(H ₂ PO ₄ ) ₂ ), silicon dioxide, manganese carbonate, cadmium carbonate, polystyrene, melamine formaldehyde, polylactic acid Polyglycolic acid (PLGA), polylactic acid (PLA), or a combination thereof.

In a preferred embodiment, the polysaccharide has an opposite electrical property after dissociation from the peptide; more specifically, the polysaccharide has the opposite effect after dissociation from the peptide in an aqueous solution (pH 7 to 8) Electrical. In a possible embodiment, the polysaccharide has a dissociation constant pK _b of 7.5 to 12. In one possible embodiment, the peptide dissociation constant pK _a of 3 to 5.

While not wishing to be bound by any theory, the present invention contemplates that when a low molecular weight of the polysaccharide and the peptide are used, there is no excessive aggregation and precipitation between the polysaccharide and the peptide, and thus it is not necessary to alternate The polysaccharide and the peptide are reacted with the porous particles, so that a cumbersome purification step can be omitted.

In a preferred embodiment, the polysaccharide and the peptide have a weight average molecular weight of not more than 5,000 Daltons; in a more preferred embodiment, the weight average molecular weight of the polysaccharide and the peptide are both Between 1,000 and 3,500 Daltons (including 1,000 Daltons and 3,500 Daltons).

In a possible embodiment, the polysaccharide and the peptide are linked to each other as a glycopeptide. Preferably, the bond is: a covalent bond, a hydrogen bond, or an ionic bond; preferably, the covalent bond is an amide bond.

In a possible embodiment, the polysaccharide is reacted with the peptide and a linker molecule to form the glycopeptide; wherein the linker molecule is: 1-ethyl-(3-dimethylaminopropyl)carbonyl Diimine (EDC), N,N-Dicyclohexylcarbodiimide, Sulfo-NHS (N-Hydroxysulfosuccinimide sodium salt)/1- Ethyl-(3-dimethylaminopropyl)carbodiimide, or a combination thereof. In a preferred embodiment, the polysaccharide and the linking molecule, and/or the peptide and the linking molecule are respectively linked by a bond; preferably, the bond is: a covalent bond, Hydrogen bond, or ionic bond.

In a preferred embodiment, the glycopeptide has a weight average molecular weight of no greater than 10,000 Daltons; in a more preferred embodiment, the glycopeptide has a weight average molecular weight of between 2,000 and 7,000 Daltons. Between (including 2,000 Daltons and 7,000 Daltons). In a possible embodiment, the polysaccharide is: chitin, trimethyl chitosan, cationic starch, or a combination thereof. In a possible embodiment, the peptide is: polyglutamic acid, polyaspartic acid, or a combination thereof.

In the spirit of the present invention, one skilled in the art can also select two polyelectrolyte molecules which will have different electrical properties after dissociation, and are not limited to the polysaccharides or peptides specifically taught by the present invention. The properties claimed in the present invention are equally achieved as long as the selected polyelectrolyte molecule has a suitable molecular weight as taught by the present invention. For example, in addition to the aforementioned polysaccharides, those skilled in the art may also employ: polyhistidine, polylysine, polyarginine, polyornithine, or polyallylamine; in addition to the aforementioned peptides, the prior art Persons can also use: polyacrylic acid, polymethacrylic acid, hyaluronic acid, or anionic strach.

In a possible embodiment, the method of the present invention further comprises the step of filling the porous material with a first active ingredient. In another possible embodiment, the method of the present invention further comprises the step (C) of dissolving the core; In this possible embodiment, the method of the present invention further comprises the step of filling the first active ingredient in the interior space defined by the shell. The type of the first active ingredient is, for example, but not limited to, Doxorubicin, folic acid, bovine serum albumin, insulin, or a combination thereof. In a possible embodiment, the second active ingredient is further grafted onto the polysaccharide and/or the peptide before the step (B). The type of the second active ingredient is, for example, but not limited to, a dye, a metal, an antibody, a receptor, or a combination thereof, and there are many different applications.

Such dyes such as fluorescent dyes include, but are not limited to, Fluorescein isothiocyanate (FITC), Rhdamine B (Rh-B), Cy-2, Cy-3, Cy-3B, Cy-3.5, Cy-5, Cy- 5.5, Cy-7, Texas red, or indocyanine green. The metal is for example but not limited to: gold nano, nano-ferric oxide, ruthenium, osmium, or iridium. Such antibodies include, but are not limited to, anti-CD4 immunoglobulin IgG, anti-CD8 immunoglobulin IgG, anti-CD19 immunoglobulin IgG, anti-CD20 immunoglobulin IgG, anti-CD22 immunoglobulin IgG, anti-CD33 immunoglobulin IgG , anti-CD 34 immunoglobulin IgG, anti-CD 44 immunoglobulin IgG, anti-CD64 immunoglobulin IgG, anti-CD 47 immunoglobulin IgG, anti-CD70 immunoglobulin IgG, anti-CD74 immunoglobulin IgG, anti-CD 79b immunization Globulin IgG, anti-CD-105 immunoglobulin IgG, and anti-CD133 immunoglobulin IgG, anti-CD138 immunoglobulin IgG, Denosumab, or a combination thereof. The receptor includes, but is not limited to, folate receptor, HER2 receptor, emodin receptor, epidermal growth factor receptor, luteinizing hormone-releasing hormone receptor, platelet-derived growth factor receptor , G protein-coupled receptors, somatostatin receptors, benzodiazepine receptors (leukotriene receptors), or chimeric T cell receptors.

Another aspect of the invention provides a polyelectrolyte capsule. Feasibly, it is made by the method of the invention. The polyelectrolyte capsule comprises: a shell; and an inner space defined by the shell; wherein the shell comprises the polysaccharide and the peptide. The polysaccharide and the peptide are as defined in the preceding paragraph. In a preferred embodiment, the polysaccharide and the peptide are linked to each other as a glycopeptide by a covalent bond. The polysaccharides, peptides, and glycopeptides are as defined in the preceding paragraphs.

In a possible embodiment, the interior space comprises a first active ingredient. In another possible embodiment, the inner space comprises a core; the core comprises porous particles and is interconnected with the shell by electrostatic attraction; wherein the porous particles comprise a porous material. In this possible embodiment, the porous material is filled with a first active ingredient. The type of the first active ingredient is, for example, but not limited to, Doxorubicin, folic acid, bovine serum albumin, insulin, or a combination thereof. In a possible embodiment, the polysaccharide and/or the peptide are grafted with a second active ingredient. The type of the second active ingredient is, for example, but not limited to, a dye, a metal, an antibody, a receptor, or a combination thereof.

In a possible embodiment, the porous material comprises: calcium carbonate, calcium superphosphate (Ca(H ₂ PO ₄ ) ₂ ), silicon dioxide, manganese carbonate, cadmium carbonate, polystyrene, melamine formaldehyde, polylactic acid Polyglycolic acid (PLGA), polylactic acid (PLA), or a combination thereof.

In a possible embodiment, the manner in which the polyelectrolyte capsules described above are administered to an individual in need can be selected as desired. The methods of administration described above are, for example but not limited to, oral, intravenous, spray, or suppository.

Specific embodiments in the spirit of the present invention will be described in the following paragraphs. The following specific examples are for illustrative purposes only and are not intended to limit the invention. The disclosure and general knowledge based on the present invention can naturally be changed according to the needs thereof by those skilled in the art, and still belong to the spirit of the present invention.

Example 1: Preparation of a polyelectrolyte capsule of the invention.

The core in the polyelectrolyte capsule of the present invention will be prepared in this embodiment, and the core is filled with the active ingredient. The porous material used in the preparation of the core in the present embodiment is calcium carbonate, and the active ingredient is doxorubicin.

Experiment A1. Calcium carbonate particles having a radius of 1 μm.

A calcium carbonate solution (0.5 M), a sodium carbonate solution (Na ₂ CO ₃ , 0.5 M), and a water-soluble starch solution (0.25 wt%) were prepared. The aforementioned calcium carbonate solution was mixed with the aforementioned water-soluble starch solution, and placed in an oven for continuous stirring for 30 minutes. Next, the above sodium carbonate solution (the final molar ratio of calcium carbonate and sodium carbonate was 1:1) was quickly added, and the mixture was vigorously stirred for 10 minutes. Then, a white precipitate, calcium carbonate, was obtained by centrifugation. The aforementioned white precipitate was allowed to dry at 50 ° C overnight. The obtained calcium carbonate particles were observed by a scanning electron microscope. As shown in Fig. 1, the calcium carbonate particles obtained in this example have a uniform size (about 1 μm).

Experiment A2. Calcium carbonate particles having a radius of 5 μm.

A calcium carbonate solution (0.33 M) and a sodium carbonate solution (Na ₂ CO ₃ , 0.33 M) were prepared. The mixed solution was obtained by rapidly and sufficiently mixing the calcium carbonate solution and the sodium carbonate solution at room temperature for 30 seconds. After the shaking of the mixture was allowed to stand still, the mixture was allowed to stand at room temperature (25 ° C) for 20 minutes to gradually form desired calcium carbonate particles. Then, the obtained calcium carbonate particles were washed with water and dried at 50 ° C overnight. The obtained calcium carbonate particles were observed by a scanning electron microscope. As shown in Fig. 2, the calcium carbonate particles obtained in this example have a uniform size (about 5 μm).

Experiment B1. Calcium carbonate particles filled with doxorubicin.

A suspension (10 mg) of calcium carbonate particles having a radius of 1 μm was obtained and mixed with doxorubicin (1 mg/mL, 2 mL) to prepare a mixed solution. The mixture was continuously stirred at room temperature for several hours (1 to 16 hours) in a dark room. Next, the calcium carbonate particles (which were filled with doxorubicin) were centrifuged, washed with water, and dried at 50 °C. The doxorubicin-filled calcium carbonate particles were observed by Fourier transform infrared spectroscopy (FT-IR), and the spectra of unfilled calcium carbonate particles and doxorubicin were compared. As a result, as shown in FIG. 3, the map of the calcium carbonate particles filled with doxorubicin has calcium carbonate particles, respectively. The peak type unique to the sub-and doxorubicin (indicated by the circle in the figure) indicates that the doxorubicin has been successfully filled in the calcium carbonate particles.

In, CaCO _3-Dox peak in Figure 3 is: 3312.62,2935.81,1403.38,1283.26,1201.96,1018.55,873.91,744.87; CaCO ₃ peak is: 1401.57,1088.61,874.31,745.20; peak of DOX: 3316.96,2934.99 , 1760.65, 1615.51, 1580.04, 1525.09, 1413.08, 1282.80, 1234.82, 1211.34, 1138.89, 1071.50, 969.15, 950.49, 912.24, 870.66, 846.13, 794.19, 761.28, 721.67, 709.49, 687.69, 600.25, 582.97.

Further, during the period in which doxorubicin is mixed with the suspension of the aforementioned calcium carbonate particles, the doxorubicin embedding rate is calculated by the following formula:

Doxorubicin embedding rate = ((Wt-Wf) / Wt) × 100%

- Wt: amount of doxorubicin initially used (measured by a spectrophotometer at a wavelength of 480 nm).

- Wf: The amount of doxorubicin remaining in the solution after the reaction of doxorubicin with calcium carbonate particles (measured by a spectrophotometer at a wavelength of 480 nm).

The doxorubicin embedding rate of 1 hour and 16 hours of mixing is shown in Figure 4. As can be seen from the data in Figure 4, the doxorubicin embedding rate can reach 97% at a mixing time of 16 hours.

Experiment B2, calcium carbonate particles filled with folic acid.

A suspension (10 mg) of calcium carbonate particles having a radius of 1 μm was obtained and mixed with folic acid (1 mg/mL, 2 mL) to prepare a mixed solution. The mixture was continuously stirred at room temperature for 1 hour in a dark room. The folic acid-filled calcium carbonate particles were then obtained following the procedure in the above paragraph B1 of the experiment. The folic acid-filled calcium carbonate particles were observed by Fourier transform infrared spectroscopy (FT-IR), and the maps of unfilled calcium carbonate particles and folic acid were compared. As a result, as shown in Fig. 5, the map of the calcium carbonate particles filled with folic acid has a peak shape unique to calcium carbonate particles and folic acid (indicated by a circle in the figure), indicating that the folic acid has been successfully filled in the calcium carbonate particles.

Experiment B3. Calcium carbonate particles filled with bovine serum albumin.

A suspension (10 mg) of calcium carbonate particles having a radius of 5 μm was obtained and mixed with bovine serum albumin (BSA, 10 mg/mL, 2 mL) to prepare a mixed solution. The mixture was continuously stirred at room temperature for 1 hour in a dark room. Next, the calcium carbonate particles filled with bovine serum albumin were obtained by following the procedure in the above paragraph B1 of the experiment. The calcium carbonate particles filled with bovine serum albumin were observed by Fourier transform infrared spectroscopy (FT-IR), and the maps of unfilled calcium carbonate particles and bovine serum albumin were compared. As a result, as shown in Fig. 6, the map of calcium carbonate particles filled with bovine serum albumin has a peak shape unique to calcium carbonate particles and bovine serum albumin (indicated by a circle in the figure), indicating that it has been successfully filled in calcium carbonate particles. Bovine serum albumin.

In Fig. 6, the peak values of BSA-CaCO ₃ are: 3319.99, 2296.73, 5122.45, 1795.17, 1651.81, 1392.94, 1157.00, 1081.27, 1023.70, 871.74, 848.12, 744.70, 712.32; the peak values of BSA are: 3284.20, 2296.37, 1644.71, 1515.92. , 1453.74, 1393.71, 1241.98, 1080.77; the peak values of CaCO ₃ are: 3355.54, 1764.17, 1403.68, 1152.72, 1081.49, 1023.54, 849.31, 745.30, 712.79.

Experiment B4, insulin-filled calcium carbonate particles.

A suspension (10 mg) of calcium carbonate particles having a radius of 1 μm was obtained and mixed with insulin (10 mg/mL, 2 mL, a 0.1 N aqueous sodium hydroxide solution) to prepare a mixed solution. The mixture was continuously stirred at room temperature for 2 hours in a dark room. Next, the insulin-filled calcium carbonate particles were obtained by following the procedure in the above paragraph B1 of the experiment. The insulin-filled calcium carbonate particles were observed by Fourier transform infrared spectroscopy (FT-IR), and the maps of unfilled calcium carbonate particles and insulin were compared. As a result, as shown in (A) of FIG. 7 and (B) of FIG. 7, the map of the calcium carbonate particles filled with insulin has a peak shape unique to calcium carbonate particles and insulin (indicated by a circle in the figure), and the display has been successful. The calcium carbonate particles are filled with insulin.

In Fig. 7 (A) and Fig. 7 (B), the peaks of the insulin-filled calcium carbonate particles are: 3395.58, 2510.06, 1652.99, 1403.52, 1153.02, 1085.79, 1022.81, 873.85, 745.43, 712.69; peaks of calcium carbonate particles. They are: 2512.07, 1975.36, 847.88, 871.54, and 712.06; the peak values of insulin are: 3287.81, 2960.81, 1645.15, 1514.94, 1451.51, 1396.00, 1238.65, 1173.03, 1127.06, 877.64.

Experiment C, Preparation of glycopeptides.

Mixing an aqueous solution of chitin (200 mg / 4 mL, molecular weight of about 2,500 Daltons) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC, 128.5 mg, 0.67 mmol) and Stir well. Next, a solution of sodium polyglutamate sodium salt (200 mg, molecular weight of about 1,308 Daltons) was added, and the resulting mixture was stirred at room temperature for 24 hours. Then, the mixture was dialyzed against a dialysis membrane (Spectra/Por molecular porous membrane, cut-off: 5,000) for 48 hours. After dialysis, the resulting glycopeptide was dried by freeze drying.

In order to be able to detect the position of the polyelectrolyte capsule of the present invention, a fluorescent dye (Fluorescein isothiocyanate (FITC) or Rhdamine B (Rh-B)) was grafted onto the obtained glycopeptide. First, 200 mg of the aforementioned glycopeptide was dissolved in 50 mL of water to obtain an aqueous glycopeptide solution, and the pH of the aqueous glycopeptide solution was controlled to be about 9. Next, a FITC methanol aqueous solution (50 mg of FITC dissolved in 25 mL of an aqueous methanol solution, methanol:water volume ratio = 1:1) was obtained, and the pH was maintained at about 9. The aqueous glycopeptide solution and the aforementioned aqueous solution of FITC methanol were then mixed overnight in the dark. Next, methanol was removed by vacuum concentration, and dialyzed against a dialysis membrane (Spectra/Por molecular porous membrane, cut-off: 1,000) for 48 hours to obtain a final product, and the final product (100 mg) was freeze-dried.

The method of grafting Rh-B onto the obtained glycopeptide is as follows. An aqueous solution of Rh-B (25 mg of Rh-B dissolved in 25 mL of water) was taken and maintained at a pH of about 9. Mixing the aforementioned aqueous glycopeptide solution and the aforementioned Rh-B aqueous solution in the dark night. Next, dialysis was carried out by a dialysis membrane (Spectra/Por molecular porous membrane, cut-off: 1,000,) for 48 hours to obtain a final product, and the final product (80 mg) was freeze-dried.

Experiment D1, linking glycopeptides and calcium carbonate particles.

An aqueous solution of glycopeptide grafted with FITC (2 mL, 1 mg/mL, a 0.15 M aqueous NaCl solution) was mixed with calcium carbonate particles (10 mg, 1 μm or 5 μm) which were not filled with the active ingredient for 1 hour in the dark. Next, it is washed in the dark, centrifuged, and the product is dried in a vacuum. The resulting product was observed under a confocal microscope. As shown in (A) and (B) of Fig. 8, a fluorescing sphere was observed in the visual field, and it was found that the glycopeptide was surely bonded to the surface of the calcium carbonate particles to obtain the polyelectrolyte capsule of the present invention.

Similarly, the aqueous solution of the glycopeptide grafted with FITC and the calcium carbonate particles filled with doxorubicin (refer to the above experiment B1, 1 μm) and the calcium carbonate particles filled with folic acid (please refer to the above experiment B2, 1 μm), and the filled cattle. The calcium carbonate particles of serum albumin (refer to the above experiment B3, 1 μm or 5 μm) and the insulin-filled calcium carbonate particles (refer to the above experiment B4, 1 μm) were connected, and the obtained product was observed by a confocal microscope. As shown in Fig. 9, Fig. 10, Fig. 11 (A) and (B), and Fig. 12, a fluorescing sphere was observed in the visual field, and it was found that the glycopeptide was indeed attached to the surface of the calcium carbonate particle. The polyelectrolyte capsule of the present invention.

Experiment D2, a microcapsule (micelle) having a glycopeptide as a shell.

Obtaining the polyelectrolyte capsule of the present invention (the linkage of the glycopeptide to the doxorubicin-filled calcium carbonate particles), and immersing it in the EDTA/0.1M HCl solution, after the HCl is infiltrated into the polyelectrolyte capsule to dissolve the calcium carbonate. That is, the microparticles of the present invention having a glycopeptide as a shell are obtained (the calcium phosphate core is no longer in the microcell, but the doxorubicin is still retained). Similarly, the polyelectrolyte capsules of the present invention which are not filled with the active ingredient can be used, and after the microcapsules are prepared by dissolving the calcium carbonate core, the active ingredients are filled into the prepared microcell particles.

It can be observed from the comparison of (A) and (B) in Fig. 13 that HCl dissolves calcium carbonate in the polyelectrolyte capsule to generate carbon dioxide. Since the polyelectrolyte capsule of the present invention has a dense glycopeptide shell, the polyelectrolyte capsule cannot be rapidly leaked out in the polyelectrolyte capsule, and the glycopeptide shell is pushed outward to expand the polyelectrolyte capsule of the present invention ( The particle size is expanded from about 1 μm to 10 μm).

Example 2: Characterization of the polyelectrolyte capsule of the present invention

Experiment E. Analysis of the active ingredient release rate of the polyelectrolyte capsule of the present invention.

This experiment simulates the release rate of the active ingredient filled in the polyelectrolyte capsule when the polyelectrolyte capsule of the present invention is orally administered. According to the study, when there is food in the stomach, the pH of the stomach environment is about 1.0 to 2.0, and the pH of the stomach environment on an empty stomach is about 2.5 to 3.7. In addition, the pH values of the duodenum, jejunum, and proximal ileum were approximately 6.0 to 6.6 and 6.6 to 7.0, respectively, and the pH of the ileal and intestinal epithelial cells was approximately 7.4. According to this, In this experiment, the polyelectrolyte capsule of the present invention (the doxorubicin-filled polyelectrolyte capsule prepared in Experiment B1) was tested for doxorubicin release rate in the environment of pH 1.4 and 7.4, and was filled with doxorubicin-containing carbonic acid. Calcium particles were used as a control group. In detail, after the experimental drug was administered orally to the patient, the drug sequentially entered the stomach (the drug was held in the stomach for 2 hours) and the time sequence of the intestine, and the release rate of the active ingredient was measured (such as the following formula). ).

The experimental results are shown in Figure 14 and Table 1 below. The release rate of doxorubicin-loaded calcium carbonate particles in the environment of pH 1.4 can reach nearly 60% (reaction time 120 minutes), and then enter the environment after pH 7.4. The rate is also roughly 60%. The polyelectrolyte capsule of the present invention has a release rate of only about 20% in a pH of 1.4 at a reaction time of 120 minutes, and gradually reaches a release rate of 60% in a pH of 7.4. The results of this experiment show that the polyelectrolyte capsule of the present invention can smoothly pass the erosion of gastric acid and release the active ingredient in the intestinal tract.

Table 1: Doxorubicin release rate (%)

In summary, the polyelectrolyte capsule of the present invention has the advantage of being simple to prepare and can be used as a pharmaceutical carrier. In particular, it can be seen from the above-described release profiles under different pH environments that the polyelectrolyte capsules of the present invention protect the active ingredient from gastric acid and release the active ingredient after reaching the neutral environment.

Claims

A method for preparing a polyelectrolyte capsule, comprising the steps of:

(A) obtaining porous particles comprising a porous material;

(B) mixing the particles with a polysaccharide and a peptide to obtain core-shell particles; wherein the core of the core-shell particle is the porous particle, and the shell of the core-shell particle comprises the polysaccharide and the peptide;

Wherein the polysaccharide and the peptide have a weight average molecular weight of not more than 5,000 Daltons.
The production method according to claim 1, wherein the polysaccharide and the peptide each have a weight average molecular weight of between 1,000 and 3,500 Daltons.
The production method according to claim 1, wherein the polysaccharide and the peptide are linked to each other by a covalent bond.
The production method according to claim 1, wherein the polysaccharide has a dissociation constant pK b of from 7.5 to 12.
The production method according to claim 1, wherein the polysaccharide is chitin, trimethyl chitosan, cationic starch, or a combination thereof.
The production method according to claim 1, wherein the peptide solution with a dissociation constant pK a of 3 to 5.
The production method according to claim 1, wherein the peptide is polyglutamic acid, polyaspartic acid, or a combination thereof.
The preparation method according to claim 1, wherein the porous material comprises: calcium carbonate, calcium superphosphate, silicon dioxide, manganese carbonate, cadmium carbonate, polystyrene, melamine formaldehyde, polylactic acid polyglycolic acid, poly Lactic acid, or a combination thereof.
The production method according to claim 1, wherein the porous active material is further filled with the first active ingredient before the step (A).
The production method according to any one of claims 1 to 9, wherein the step (B) further comprises a step (C): (C) dissolving the core.
The production method according to claim 10, wherein the polyelectrolyte capsule comprises an inner space defined by the shell, wherein the preparation method further comprises filling the inner space with a first active ingredient.
The preparation method according to any one of claims 9 or 11, wherein the first active ingredient comprises: doxorubicin, folic acid, bovine serum albumin, insulin, or a combination thereof.
The preparation method according to claim 1, wherein a second active ingredient is further grafted onto the polysaccharide and/or the peptide before the step (B).
The preparation method according to claim 13, wherein the second active ingredient comprises: a dye, a metal, an antibody, a receptor, or a combination thereof.
A polyelectrolyte capsule comprising:

Shell; and

An internal space defined by the shell;

Wherein the shell comprises a glycopeptide; wherein the glycopeptide comprises: a polysaccharide and a peptide; wherein the polysaccharide and the peptide are linked to each other via a bond;

Wherein the glycopeptide has a weight average molecular weight of no greater than 10,000 Daltons.
The polyelectrolyte capsule according to claim 15, wherein the glycopeptide has a weight average molecular weight of 2,000 to 7,000 Daltons.
The polyelectrolyte capsule according to claim 15, wherein the polysaccharide has a dissociation constant pKb of 7.5 to 12.
The polyelectrolyte capsule according to claim 15, wherein the polysaccharide is: chitin, trimethyl chitosan, cationic starch, or a combination thereof.
Polyelectrolyte capsule according to claim 15, wherein the peptide solution with a dissociation constant pK a of 3 to 5.
The polyelectrolyte capsule according to claim 15, wherein the peptide is polyglutamic acid, polyaspartic acid, or a combination thereof.
The polyelectrolyte capsule of claim 15 wherein said interior space comprises a first active ingredient.
The polyelectrolyte capsule according to claim 15, wherein the inner space contains a core, the core comprises porous particles and is interconnected with the shell by electrostatic attraction; wherein the porous particles comprise a porous material.
The polyelectrolyte capsule according to claim 16, wherein the porous material comprises: calcium carbonate, calcium superphosphate, silicon dioxide, manganese carbonate, cadmium carbonate, polystyrene, melamine formaldehyde, polylactic acid polyglycolic acid, Polylactic acid, or a combination thereof.
The polyelectrolyte capsule according to claim 22, wherein the porous material is filled with a first active ingredient.
The polyelectrolyte capsule according to any one of claims 21 or 24, wherein the first active ingredient comprises: doxorubicin, folic acid, bovine serum albumin, insulin, or a combination thereof.
The polyelectrolyte capsule according to claim 15, wherein the shell is grafted with a second active ingredient; wherein the second active ingredient comprises: a dye, a metal, an antibody, a receptor, or a combination thereof.
The polyelectrolyte capsule according to claim 15, wherein the bond is a covalent bond, a hydrogen bond, or an ionic bond.
The polyelectrolyte capsule according to claim 27, wherein the covalent bond is an amide bond.