CN1887273A - Prepn process of polysaccharide vitreous particle - Google Patents

Abstract

The invention relates to a method for preparing polysaccharide vitreous particles in the technical field of biomedicine. The present invention comprises the following steps: ① dissolving polyethylene glycol, polyethylene oxide or polypyrrolidone and polysaccharide in water to form a solution, and mixing them at a ratio greater than 1:1 by weight under low temperature conditions. ②Add bioactive substances to it, or add together with polysaccharides and polyethylene glycol; ③Freeze the emulsion that has completed step ②; ④Freeze-dry the sample that has completed step ③; The sample is washed with a solvent to remove the PEG, PEO or PVP continuous phase to obtain polysaccharide dispersed phase particles. The present invention completely avoids the use of organic solvents, high shear force, interface, and cross-linking agents, etc., and maintains the activity of biologically active substances for a long time under mild conditions. Greatly reduce the cost of preservation and improve the curative effect. The polysaccharide vitreous body prepared at the same time has small particle size and high activity, so when it is prepared into other dosage forms such as sustained-release microspheres, burst release and incomplete release can be reduced.

Description

Method for preparing polysaccharide vitreous particles

technical field

The invention relates to a preparation method in the technical field of biomedicine. In particular, a method for preparing polysaccharide vitreous particles.

Background technique

Since gene recombination technology has been used for the expression and production of therapeutic proteins for more than 20 years, so far, more than 30 protein drug products have been put into clinical use, and nearly 200 are in the process of approval and research and development, and a number of such as Amgen ( Amgen), Gene Technology (Genentech) and other new large pharmaceutical companies. Compared with the rapid development of protein macromolecular drugs, their dosage form technology progresses slowly. On the one hand, protein macromolecular drugs are not absorbed orally and have a short half-life in vivo, requiring injection; on the other hand, many hormone and cytokine protein drugs have a long treatment cycle, and long-term and frequent injections become necessary, which also affects major cause of patient compliance. The development of dosage forms of sustained-release protein drugs is due to the loss of activity during the preparation of microparticles, such as the W/O/W method. It is imperative to develop and prepare protein particles with active protection. Dextran and PEG are commonly used as lyoprotectants for protein drugs and as reagents for separating and purifying proteins. Hennink used modified dextran to prepare protein sustained-release microspheres, but the author used strong oxidants such as potassium persulfate, ammonium persulfate, etc., and polymer initiators to cross-link the dextran molecules, which inevitably did not match The protein reacts, resulting in inactivation of the protein or increased immunogenicity. So far, there is no report on the preparation of microparticles of polymers in an aqueous environment without cross-linking or curing agents.

[Hennink.W.E.and Franssen.O.PROCESS FOR THE PREPARATION OF A CONTROLLED RELEASESYSTEM.Publication No.: WO/1998/022093 International Application No.: PCT/NL1997/000625], (Hennink.W.E.and Franssen.O. a preparation method of a controlled release system, publication number.: WO/1998/022093. International application number: PCT/NL1997/000625), the patent reported Aqueous-aqueous two-phase system for the preparation of polysaccharide particles. However, they use polysaccharides that can be cross-linked, and the cross-linking groups are chemically modified. This will not only change the properties of the polysaccharide itself, but may increase the immunogenicity of the polysaccharide. At the same time, these cross-linking groups may also cross-link with proteins and polypeptides, because the cross-linking groups have no special selectivity. We avoided the use of crosslinking to destabilize aqueous-aqueous emulsions to prepare polysaccharide vitreous bodies.

Patents in prior art [CHEN, Li; ZHU, Hua; JIN, Tuo THE PREPARATION METHOD OF A STABLE POLYMER AQUEOUS PHASE-AQUEOUS PHASE EMULSION AND ITSUSE, Publication Number International: WO/2002/000778 Application No.: PCT/CN2001/ 001033] (Chen Li; Zhu Hua; the preparation and application publication number of Jintuo stable polymer water phase-water phase emulsion is, WO/2002/000778 international application number PCT/CN2001/001033), this patent discloses a Novel stable emulsion (polymer water phase-water phase emulsion) and its preparation method and application in pharmacy and biotechnology. The fundamental difference between this emulsion and conventional emulsions is that its dispersed phase and continuous phase are both aqueous solutions. That is, by introducing the polymer electrolyte into the polymer hydrophilic two-phase system (aqueous two-phase system) to generate a diffuse electric double layer, a unique material system has been developed: stable without continuous stirring or (for the dispersed phase) instant Curing polymer water-water emulsion. This system was used in the preparation of sustained-release microspheres for protein macromolecular drugs, and ideal results were obtained. However, some protein molecules may interact with polyelectrolytes, causing aggregation.

content of the invention

The purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing polysaccharide vitreous particles. The surface of the prepared particles is smooth and round, the uniformity is good, the particle size has no adhesion, and there is no organic solvent, no cross-linking agent, polymer initiator and surfactant, especially polymer electrolyte surfactant. To avoid these effects on the therapeutic effect of the drug.

The present invention is achieved through the following technical solutions: the present invention is under the condition of -2°C-4°C, two or more mutually indescribable water-soluble polymers, under external conditions such as homogenization, Voterx, ultrasonic The emulsion is formed under the action of mechanical stirring, etc., and then freeze-dried and the continuous dispersed phase is removed to obtain polysaccharide glass particles. No need to use organic phase (or oil phase) for emulsification, no need to use crosslinking agent, curing agent, surfactant, and there is no interface with strong interfacial tension such as water-oil, water-gas.

The present invention specifically comprises the following steps:

① Dissolve polyethylene glycol (PEG), polyethylene oxide (PEO) or polypyrrolidone (PVP) and polysaccharides in water to form a solution. The ratio is mixed, and under the action of external force, an emulsion is formed;

② Add biologically active substances to it, or add polysaccharides and polyethylene glycol together, or add no drugs;

③ freezing the emulsion that has completed step ②;

④ freeze-drying the sample that has completed step ③;

⑤ Wash the sample after step ④ with a solvent to remove the PEG, PEO or PVP continuous phase to obtain polysaccharide dispersed phase particles.

The PEG, PEO or PVP has a molecular weight of 1,000-30,000.

The concentration of said PEG, PEO or PVP is between 0.2% and 50%.

The polysaccharide has a molecular weight of 50,000-500,000.

The concentration of the polysaccharide is 2%-20%.

The polysaccharides are dextran or dextran, starch, cellulose and its derivatives, agarose and water-soluble polymer polysaccharides.

The PEG, PEO or PVP is used to remove PEG, PEO or PVP, and the organic solvent of the continuous phase is a solvent that dissolves PEG but does not dissolve polysaccharides.

The polysaccharide dispersed phase particles contain buffer substances, salts and small molecular sugar substances.

The buffer substance is Mg(OH) ₂ , ZnCO ₃ , and MgCO ₃ , the salt is one of Zn(AC) ₂ , ZnCl ₂ , CaSO ₄ , and MgSO ₄ ; the small molecule sugar is trehalose, sucrose , glucose, sorbitol, mannitol in one.

The polysaccharide dispersed phase particles can be used to microencapsulate therapeutic substances with fragile structures—such as proteins, polypeptides, genetic materials, antibodies, vaccines, viruses, and liposomes, which can be loaded in polysaccharide particles;

The polysaccharide dispersed phase particles can be used in sustained-release microsphere dosage forms and inhalation dosage forms of therapeutic substances carried by them;

In the sustained-release microsphere dosage form, the polysaccharide particles loaded with therapeutic substances are dispersed in the matrix of degradable polymer microspheres; the colostrum (S/O) is formed by dispersing the polysaccharide particles in the degradable polymer solution, and then Dispersed in the water phase to form double emulsion, that is, the method of solid-in-oil-oil-in-water (S/O/W), or by dispersing polysaccharide vitreous particles in an oil-soluble degradable polymer solution to form colostrum (S/O ), and then dispersed in another oil phase to form a double emulsion, that is, a solid-in-oil-oil-in-oil (S/O/O) method.

The inhalation dosage form is the polysaccharide itself loaded with therapeutic substances, and its particle size is 1 μm-5 μm.

1. Preparation of blank polysaccharide vitreous body

1. Preparation of PEG and Polysaccharide Aqueous Solutions

Prepare 10% PEG and 10% polysaccharide aqueous solution with ultrapure water, accurately weigh 10 grams of PEG and 10 grams of polysaccharide water, add 90 grams of water to a 100 ml beaker, and then place the two beakers on a heated magnetic Stir for 30 minutes, wait until the PEG and polysaccharide are all dissolved, take it out and cool it for later use.

2. Preparation of PEG and polysaccharide low temperature water phase-water phase emulsion

Under the condition of 0°C-4°C, mix the polysaccharide and PEG aqueous solution in 1. according to the volume ratio of 1:5, 1:10, 1:20, and 1:40 in the vial and vortex for 30s-60s to form Water phase-water phase emulsion; or weighed according to the mass ratio of polysaccharide and PEG of 1:5, 1:10, 1:20, 1:40 in a vial and then mixed with water to dissolve it to form a water phase-water phase emulsion.

3. Preparation of PEG and polysaccharide low temperature water phase-water phase emulsion by freezing

The prepared PEG and polysaccharide low-temperature water-water phase emulsion are frozen overnight in the refrigerator, or quick freezing will not affect the size of the vitreous body.

4. Freeze-dry the sample completed in step 3 in a vacuum freeze dryer;

5. The sample that completed step 4 was washed three times with dichloromethane to remove the PEG continuous phase to obtain polysaccharide dispersed phase particles.

2. Preparation of drug-loaded polysaccharide vitreous

1. Preparation of PEG and Polysaccharide Aqueous Solutions

All were prepared according to the blank polysaccharide vitreous body preparation method.

2. Under the condition of 0°C-4°C, mix the drug and polysaccharide solution evenly, or mix the drug, PEG and polysaccharide aqueous solution together to prepare drug-loaded PEG and polysaccharide low-temperature water-water phase emulsion.

3. Low-temperature water-water emulsion of frozen polysaccharides, drugs and PEG

4. Freeze the prepared polysaccharide, drug and PEG low temperature water phase-water phase emulsion overnight.

5. Then, according to the method for preparing blank polysaccharide vitreous bodies 4 and 5, obtain drug-loaded polysaccharide vitreous bodies.

The drug-loaded polysaccharide vitreous body prepared by the present invention can be directly used in inhalation dosage forms to protect fragile biological agents such as proteins and polypeptides; colostrum (S/O) is formed by dispersing polysaccharide particles in a degradable polymer solution, and then Dispersed in the water phase to form double emulsion, that is, the method of solid-in-oil-oil-in-water (S/O/W), or by dispersing polysaccharide vitreous particles in an oil-soluble degradable polymer solution to form colostrum (S/O ), and then dispersed in another oil phase to form a double emulsion, that is, a solid-in-oil-oil-in-oil (S/O/O) method.

The present invention completely avoids the use of organic solvents, high shear forces, interfaces, and cross-linking agents, etc., and loads biologically active substances such as proteins, polypeptides, vaccines, DNA, RNA, viruses, and liposomes under mild conditions. Polysaccharide vitreous, able to maintain activity for a long time. Greatly reduce the cost of preservation and improve the curative effect. The polysaccharide vitreous body prepared at the same time has small particle size and high activity, so when it is prepared into other dosage forms such as sustained-release microspheres, burst release and incomplete release can be reduced.

Detailed ways

Example 1: Preparation of blank polysaccharide vitreous body

1. At 0°C-4°C, prepare 10% Dex70,000 and 10% PEG8,000 solutions; take 0.500g Dex70,000; 2.500g PEG8,000, that is, 1:5 (w/w) vortex Mix well for 30s-60s to form a water phase-water phase emulsion, pre-freeze in a refrigerator at -26°C for one night, then dry in vacuum for 24 hours, take 3ml-4ml of dichloromethane with a dropper and drop it into a vial, vortex for 5min, Centrifuge at 12000RPM for 5 minutes, remove the supernatant, go back and forth three times, evaporate dichloromethane in vacuum drying, collect particles, take a small amount and observe under a microscope, or suspend it in dichloromethane and measure its particle size with a PCS particle size scattering instrument. path. The particle size is 2μm-10μm, and the average particle size is 5.7μm.

2. Under the condition of 0°C-4°C, prepare 10% Dex70,000 and 10% PEG8,000 solution; take 0.250g Dex70,000; 2.500g PEG8,000, that is, 1:10 (w/w), Vortex for 30s-60s to mix well to form a water phase-water phase emulsion, pre-freeze in a refrigerator at -26°C overnight, then dry in vacuum for 24 hours, use a dropper to take 3ml-4ml of dichloromethane and drop it into a vial, vortex for 5min, Centrifuge at 12000RPM for 5min, remove the supernatant, go back and forth three times, evaporate the dichloromethane in vacuum drying, collect the particles, take a small amount and observe it under a microscope, or suspend it in dichloromethane and measure it with a PCS particle size scattering instrument particle size. The particle size is 1μm-5μm, and the average particle size is 2.652μm.

3. Prepare 10% Dex70,000 and 10% PEG8,000 solution at 0°C to 4°C; take 0.125g Dex70,000; 2.500g PEG8,000, that is, 1:20 (w/w), Vortex for 30s-60s to mix well to form a water phase-water phase emulsion, pre-freeze in a refrigerator at -26°C overnight, then dry in vacuum for 24 hours, use a dropper to take 3ml-4ml of dichloromethane and drop it into a vial, vortex for 5min, Centrifuge at 12000RPM for 5min, remove the supernatant, go back and forth three times, evaporate the dichloromethane in vacuum drying, collect the particles, take a small amount and observe it under a microscope, or suspend it in dichloromethane and measure it with a PCS particle size scattering instrument particle size. The particle size is 0.5μm-5μm, and the average particle size is 1.562μm.

4. Under the condition of 0℃-4℃, prepare 8% Dex70,000 and 8% PEG8,000 solution; take 0.080g Dex70,000; 3.200g PEG8,000, that is, 1:40(w/w ), vortex for 30s-60s to fully mix to form a water phase-water phase emulsion, pre-freeze in a refrigerator at -26°C for one night, then dry in vacuum for 24 hours, take 3ml-4ml of dichloromethane with a dropper and drop it into a vial, vortex 5min, centrifuge at 12000RPM for 5min, remove the supernatant, go back and forth three times, evaporate the dichloromethane in vacuum drying, collect the particles, take a small amount to observe under the microscope, or suspend it in dichloromethane and use a PCS particle size scattering instrument Measure its particle size. The particle size is 0.3μm-5μm, and the average particle size is 309nm.

Example 2: Preparation of bovine serum albumin-loaded polysaccharide vitreous body

1. Preparation of bovine serum albumin, PEG and polysaccharide aqueous solution

Prepare 10% PEG and 10% polysaccharide aqueous solution with ultrapure water, accurately weigh 10 grams of PEG and 10 grams of polysaccharide water, add 90 grams of water to a 100 ml beaker, and then place the two beakers on a heated magnetic Stir for 30 minutes, wait for the PEG and 10% polysaccharide to dissolve completely, take it out and cool it for later use. Accurately weigh 800 mg of bovine serum albumin with an electronic balance and dissolve it in 7.2 ml of water for later use.

2. Bovine serum albumin, PEG and polysaccharide aqueous phase-aqueous phase emulsion

Under the condition of 0°C-4°C, the polysaccharide, bovine serum albumin and PEG aqueous solution of 1. were respectively 1:1:5, 1:1:10, 1:1:20, 1:1:40 according to the volume ratio Vortex in the vial for 30s-60s and mix thoroughly to form a water phase-water phase emulsion; or weigh 1:1:5, 1:1:10, 1:1:20, 1:1 according to the mass ratio of polysaccharide and PEG 1:40 in the vial, mix firstly, then dissolve in water, and then add polysaccharide and bovine serum albumin according to the weight ratio of 1:1, so as to form water phase-water phase emulsion.

3. Frozen bovine serum albumin, PEG and polysaccharide aqueous phase-aqueous phase emulsion

The bovine serum albumin, PEG and polysaccharides will be prepared to form a water phase-water phase emulsion, and some proteins that are not distributed to the polysaccharide phase will be redistributed into the polysaccharide dispersed phase during the cooling process, and frozen overnight.

4. The sample completed in step 3 is freeze-dried in a vacuum freeze dryer;

5. The sample completed in step 4 was washed three times with dichloromethane to remove the PEG continuous phase to obtain bovine serum albumin-loaded polysaccharide vitreous body.

The particle size of the bovine serum albumin-loaded polysaccharide dispersed phase vitreous body is mostly in the range of 300nm-5μm, and the obtained vitreous body is smooth and round, and the particle size distribution is uniform. The structure of the bovine serum albumin can be well protected, and the inactivation in the preparation process of the dosage form can be avoided.

Claims (8)

1, a kind of method for preparing polysaccharide vitreous particle is characterized in that, may further comprise the steps:

1. with Polyethylene Glycol, poly(ethylene oxide) or polypyrrole alkane ketone and polysaccharide wiring solution-forming soluble in water, mixed according to weight under cryogenic condition greater than 1: 1 ratio, under the effect of external force, form emulsion;

2. bioactive substance is added wherein, or add in the lump with polysaccharide, Polyethylene Glycol;

3. completing steps Emulsion 2. is freezing;

4. with completing steps sample lyophilizing 3.;

5. completing steps sample is 4. removed PEG, PEO or PVP continuous phase acquisition polysaccharide dispersion particle with solvent wash.

2, the method for preparing polysaccharide vitreous particle according to claim 1 is characterized in that, described Polyethylene Glycol, poly(ethylene oxide) or polypyrrole alkane ketone, and its molecular weight is 1,000-30,000.

3, according to claim 1 or the 2 described methods that prepare polysaccharide vitreous particle, it is characterized in that, described Polyethylene Glycol, poly(ethylene oxide) or polypyrrole alkane ketone, its concentration is 0.2%-50%.

4, the method for preparing polysaccharide vitreous particle according to claim 1 is characterized in that, described polysaccharide, its molecular weight are 50,000-500,000.

5, according to claim 1 or the 2 described methods that prepare polysaccharide vitreous particle, it is characterized in that, described polysaccharide, its concentration is 2%-20%.

According to claim 1 or the 2 described methods that prepare polysaccharide vitreous particle, it is characterized in that 6, described polysaccharide is glucosan or glucosan, starch, cellulose and derivant thereof, agarose and water soluble polymer polysaccharide.

7, according to claim 1 or the 2 described methods that prepare polysaccharide vitreous particle, it is characterized in that, described polysaccharide, its dispersion particle contains buffer substance, salt and micromolecule saccharide material.

According to claim 1 or the 2 described methods that prepare polysaccharide vitreous particle, it is characterized in that 8, described buffer substance is Mg (OH) ₂, ZnCO ₃, and MgCO ₃, salt is Zn (AC) ₂, ZnCl ₂, CaSO ₄, MgSO ₄In a kind of; The micromolecule saccharide is a kind of in trehalose, sucrose, glucose, sorbitol, the mannitol.