CN100556400C - The preparation method of the polysaccharide vitreous particle of carrying active vaccine or antibody - Google Patents
The preparation method of the polysaccharide vitreous particle of carrying active vaccine or antibody Download PDFInfo
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Abstract
What the present invention relates to is the preparation method of the polysaccharide vitreous particle of the carrying active vaccine in a kind of biological medicine technology field or antibody.Concrete steps are as follows: 1. vaccine or antibody are added polysaccharide solution; 2. prepared vaccine or antibody-polysaccharide are mixed in temperature above freezing; 3. formed mixed solution or water-water milkiness liquid cooling lyophilizing is dry; 4. resulting lyophilized powder is added the organic solution of soluble polyethylene glycol washing, removes Polyethylene Glycol, obtain being loaded with vaccine or antibody-polysaccharide vitreous particle.The vaccine and the antibody polysaccharide vitreous particle smooth surface rounding of the present invention's preparation, the granule regular without adhesion, particle diameter can be regulated and control from 0.1 μ m as required to 10 μ m.Can keep activity and therapeutic effect for a long time.
Description
Technical field
What the present invention relates to is the preparation method in a kind of biological medicine technology field.The preparation method of the polysaccharide vitreous particle of particularly a kind of carrying active vaccine or antibody.
Background technology
Immunization therapy is being played the part of important role at the prevention of infectious disease and tumor and treatment.The means of immunization therapy are nothing more than the use for human body of various antigens and antibody.No matter antibody is monoclonal or polyclone, is protein macromolecule, and antigen is quite variation then, comprises cell, virus, albumen, polypeptide and genetic stew.
During the nearly last ten years, with the polymer of degradable, bio-compatible is the sustained-release micro-spheres of substrate has success on micromolecule chemicals and polypeptide drugs application, this class sustained-release micro-spheres can make polypeptide antigen long-time (or multiple-pulse) release in vivo, and produce antibody response and other immunne response of continuing also has report more.By nanometer or small carrier antigen is transported to the APC endochylema, thereby the work that inducing cell immunity, particularly CTL are replied has also attracted suitable research input.How protein macromolecule antigen, antibody are encapsulated in the delivery system of this class for the peptide molecule success on this basis, making it to look like to be released to target position specific in blood circulation or the body with natural structure is problem to be solved by this invention.
For the natural structure picture that keeps protein macromolecule in the degradable macromolecule dosage form, many researcheres were done good try.Such as, in the past few decades in, saw various reports in the document about the method that improves the stability of protein in the microcapsule packet procedures.But these methods can only solve one or a part of problem, and can not take into account for other problems, even cause the problem that makes new advances.Also some method only is applicable to specific a kind of protein.The different mutual contradictions that seem of research field that also have some reports also self to be paid close attention to owing to researcher.The explanation of giving an example, for unique like product one controlled-release recombinant human class growth hormone (rhGH) on the market, its stability by and zinc form complex and realize.And form complex with zinc is rhGH natural form in vivo.When this zinc complexation preparation method is used for another kind of albumen, as erythropoietin (EPO), accumulative ratio takes place protein molecular can cause to cause immunogenic misgivings up to 40%.For avoiding albumen in the microcapsule packet procedures, to be out of shape inactivation because of removing organic solvent; scientist is made as solid granular with protein molecular and saccharide, inorganic salt or other protective agents in advance; thereby take a kind of solid dispersion at oil phase; (promptly oil wraps solid-water bag oil, method microcapsule bag protein drug S-O-W) further being dispersed in water.These protein stabilizedization adjuvants cause prominent releasing usually owing to its high-dissolvability produces strong osmotic pressure.Such as, when the high sulfate of dissolubility is used for improving EPO when the element of microcapsule packet procedures is stable, prominent releasing up to 55% of accumulated dose.
[Cleland, J.L., Jones J.S., Stable formulations of recombinant humangrowth hormone and interferon-gama for microencapsulation inbiodegradable microspheres.Pharm.Res., 1996.13:1464-1475.] (Cleland, J.L., Jones J.S., stable recombinant human growth element and an ancient woman's ornament interferon dosage form are used for biodegradable microsphere microencapsulation. study of pharmacy, 1996, the 13rd phase, the 1464-1475 page or leaf); Cleland and Jones have studied in W-O-W (Water-In-Oil-oil-in-water) and S-O-W microencapsulation process various adjuvants to the protective effect of recombinant human growth hormone and interferon, find that mannitol and trehalose can play best effect aspect this preventing that albumen from assembling in the microcapsule process.Sanchez has verified similar excipient for another kind of albumen---and the protective effect of tetanus fermentoid element found that to be identified as in the report of Cleland and Jones and can play the better preserved function to protein in the release stage under the hydrating condition to restoring the glucosan that recombinant human growth hormone and interferon do not play useful effect.As if such experiment has illustrated that monosaccharide can play the better protection effect to albumen in dry run, and macromolecular compound then plays better effect in the release stage.But, the release profiles of the glucosan of Sanchez-PLGA complex microsphere slow release formulation shows that 60% drug loading has taken place by prominent releasing.Perhaps, this prominent release is because oversize the causing of albumen-granules of accessories of direct lyophilization preparation.
In the S-O-W process, the size of the protein body of pre-preparation is very important.[Takahiro.Morita, Yuji Horikiri, Hiroshi Yamahara, Takehiko Suzuki, and Hiroyuki Yoshino.Formation and Isolation of Spherical fine protein Microparticles throughLyophilization of protein-poly (ethylene glycol) Aqueous Mixture.Pharm.Res., 17:1376-1373 (2000)], (Takahiro.Morita, Yuji Horikiri, HiroshiYamahara, Takehiko Suzuki, and Hiroyuki Yoshino. prepares the ball-type protein microsphere by freezing albumen and Polyethylene Glycol water mixed solution, study of pharmacy, 2000, the 17th phase, the 1376-1373 page or leaf); Morita find when the particulate diameter of solid protein matter when 5 microns increase to 20 microns, not only the rate of release at initial stage is double, its microencapsulation rate drops to 20% from 80%.Cleland has discussed at S-O-W and has crossed the method that the Cheng Qian dwindles protein microbeads volume size.Further pulverize the protein lyophilized powder and can not obtain diameter, grind the protein change that heat causes and the method that these powder grind to form littler size is hampered by less than 10 microns microgranule.Though the atomizing seasoning can obtain expecting the protein microbeads of size, but the heat of shear force therebetween and generation is understood owing to causing property of the intervention change that the gas liquid interface has been arranged.Spray drying method perhaps can provide enough little protein body, but high shear force and the interfacial tension between liquid-to-air near the nozzle can make albuminous degeneration.What more be far from good is, must use surfactant in spray drying or the atomizing lyophilization, and surfactant has and facilitated protein to interact with solvent in next step preparation program.Maa etc. have reported the gathering that in advance method of recombinant human growth hormone and zinc complexation was prevented protein component before spray drying.But zinc complexation meeting causes growth hormone albuminous degeneration in addition.[Takahiro.Morita, Yuji Horikiri, Hiroshi Yamahara, Takehiko Suzuki, and Hiroyuki Yoshino.Formation and Isolation of Spherical fine protein Microparticles throughLyophilization of protein-poly (ethylene glycol) Aqueous Mixture.Pharm.Res., 17:1376-1373 (2000)], (Takahiro.Morita, Yuji Horikiri, HiroshiYamahara, Takehiko Suzuki, and Hiroyuki Yoshino. prepares the ball-type protein microsphere by freezing albumen and Polyethylene Glycol water mixed solution, study of pharmacy, 2000, the 17th phase, the 1376-1373 page or leaf); The method of usefulness PEG precipitating proteins such as Motita has prepared the albumen ultramicro powder.But these microgranules still directly are under the effect of organic solvent in the microcapsule packet procedures.Because protein is not subjected to the protein of any protection and direct contact of PLGA can cause incomplete release for the adsorption on the surface, inside of macromolecular compound substrate.For fear of hydrophilic-hydrophobic separating surface, scientist once prepared the macromolecular compound microgranule with the hydrophilic diphasic system.Yet, must lean on covalency or the synchronous crosslinked action of ion to come cured granulate at emulsifying stage, thereby protein is exposed in the active reaction thing.
Number of patent application: CN02146543.6, publication number: CN1401328, title: epidemic cerebrospinal meningitis polysaccharide-protein combined vaccine, this technology with A group, the C group's epidemic cerebrospinal meningitis Neisseria gonorrhoeae capsular polysaccharide method by chemistry to be covalently bound on the effective protein proteins carrier, prepare the united vaccine formulation that contains two kinds of combined vaccines, said preparation can be used to prevent the infection of A group, C group's epidemic cerebrospinal meningitis Neisseria gonorrhoeae for the human immunity inoculation.But this technology be at be non-protein subunit vaccine, but do carrier with albumen, cause the problem of the originality of exempting from service of foreign protein itself to be difficult to avoid, and be by chemical modification, same chemical modification causes the variation of character of vaccine itself unknown.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, a kind of method of utilizing polysaccharide vitreous protection vaccine and antibody activity is provided.And become nanometer or small carrier that antigen is transported in the endochylema of APC cell, thereby inducing cell immunity, particularly CTL cell by polysaccharide vitreous protection vaccine production.Or the microsphere that is prepared into slow release keeps antigen concentration in the blood, thereby stimulates the long-term secretory antibody of plasma cell, induces humoral immunization.Local sustained release antibody mainly is rapid at the blood vessel hyperplasia of solid tumor, utilizes the local injection sustained-release micro-spheres, and specificity inhibition tumor vessel hypertrophy makes that tumor can not get enough getting nutrition and dying of hunger.
The present invention is achieved by the following technical solutions: concrete steps of the present invention are as follows:
1. vaccine or antibody are added polysaccharide solution;
2. the polyglycol solution of prepared vaccine or antibody-polysaccharide 6% or 6% above concentration solution and same concentrations is mixed being lower than under the temperature that room temperature is higher than freezing point, or with vaccine or antibody-polysaccharide 6% or 6% above concentration solution with and the polyglycol solution that contains the polyelectrolyte of 0.2-2% mix in temperature above freezing, or vaccine or antibody-polysaccharide 6% following concentration solution are mixed in temperature above freezing with the same concentrations polyglycol solution;
3. formed mixed solution or water-water milkiness liquid cooling lyophilizing is dry;
4. resulting lyophilized powder is added the organic solution of soluble polyethylene glycol washing, removes Polyethylene Glycol, obtain being loaded with vaccine or antibody-polysaccharide vitreous particle.
Described Polyethylene Glycol, its molecular weight are 1,000-30,000; Its concentration is 0.2%-50%.
Described Polyethylene Glycol, the organic solvent that is used to remove PEG continuous phase are dissolving PEG and do not dissolve the solvent of polysaccharide.
Described polysaccharide, its molecular weight are 10,000-2,000,000; Its concentration is 2%-20%.
Described polysaccharide is glucosan.
Described polyelectrolyte is sodium alginate, the electronegative polyelectrolyte of sodium carboxymethyl cellulose.
Described polysaccharide dispersion particle contains buffer substance, salt and micromolecule saccharide material.
Described buffer substance is Mg (OH)
2, ZnCO
3, and MgCO
3, salt is Zn (AC)
2, ZnCl
2, CaSO
4, MgSO
4, the micromolecule saccharide is trehalose, sucrose, glucose, sorbitol, mannitol.
Described vaccine is subunit vaccine, live-virus vaccine, deactivation viral vaccine, dead viral vaccine, nucleic acid vaccine.
Described subunit vaccine is: hepatitis A vaccine, Hepatitis B virus vaccine, hepatitis C vaccine, avian influenza vaccine, hepatitis E vaccine, AIDS vaccine, anthrax vaccine, tumor vaccine, SARS vaccine, influenza vaccines, schistosomicide vaccine, can also add the effect that immunological adjuvant such as cytokine, TAP, inorganic salt, CpG etc. play enhance immunity.
Described live-virus vaccine is Measles Vaccine, poliomyelitis vaccine, pestilence vaccine, bacillus calmette-guerin vaccine.
The percentage by weight 0.001%-50% of described subunit vaccine, the described antibody 0.001%-50% that is weight percentage, described polysaccharide percentage by weight 50-90%.
Described antibody is anti-angiogenic outgrowth antibody, the antibody of antiviral antibody, tumour antibody and cytokine antibodies or antibacterial agent.Be loaded in vaccine and antibody in polysaccharide vitreous, can keep activity and therapeutic effect for a long time.
The sustained-release micro-spheres dosage form, the polysaccharide microparticulate that is loaded with antibody, vaccine and virus is in the substrate of degradable macromolecule microsphere; Form colostrum (S/O) in the macromolecular solution, redispersion forms the i.e. method of oil bag solid-water bag oil (S/O/W) of emulsion at aqueous phase, perhaps form colostrum (S/O) by polysaccharide vitreous particle being dispersed in the molten degradable macromolecule solution of oil, redispersion forms the i.e. method of oil bag solid-water oil bag oil (S/O/O) of emulsion in another oil phase, perhaps, perhaps add in the various gels by the spray drying method for preparation microsphere.
Described macromolecule is the polyamino acid of Polyetherimide, polyester, poly-anhydride, poly-amino ester or skeleton non-peptide bond, comprising: polylactic acid, polylactic acid-polyglycolic acid and combination thereof; Comprise that also silicon is as glue, politef, polrvinyl chloride, polyethylene, polypropylene, polystyrene, polyethylene terephthalate, Merlon, Carmomer, hyaluronic acid, gelatin, collagen protein, poly-Acetic acid, hydroxy-, bimol. cyclic ester, polycaprolactone, polybutylcyanoacrylate, poly phosphazene, poly phosphate, Fibrinogen, fibrin and their combination, gel comprises: polyethylene glycol-lactic acid, the polyethylene glycol-hydroxyacetic acid, polyglycolic acid-polyethylene glycol-hydroxyacetic acid, polylactic acid-polyglycol-polylactic acid temperature-sensitive hydrogel, quick type gel of sugar and acid-sensitive type gel, their combination.
Described temperature sensitive type gel is characterized by the albumen that is stated from vaccine or antibody polysaccharide vitreous particle or the polysaccharide solution and optionally is distributed in polysaccharide and is on the thermodynamics more stable status in mutually.In addition, the temperature sensitive type gel ties up in intensification phase transformation (from liquid to the glue) process prominent the releasing that syneresis causes is taken place, and is distributed in the albumen of polysaccharide in mutually and can avoids to a great extent then that this class is prominent to be released.Described polysaccharide vitreous or can be introduced directly into the temperature sensitive type gel that is in liquid condition under the low temperature or their combination without the freeze dried polysaccharide solution that contains vaccine or antibody of emulsifying.
The microsphere dosage form of described slow release vaccine is a subcutaneous injection, plays the vaccine that long-term slow release has treatment and preventive effect.
The microsphere dosage form of described slow release antibody is the solid tumor surrounding injection, plays long-term slow release and improves solid tumor antibody concentration on every side, plays the effect of treatment tumor.
The microsphere dosage form of the cytophagic vaccine of described APC is the microsphere of 0.5 μ m-10 μ m, and subcutaneous injection or intramuscular injection can be caused cellular immunization or humoral immunization by the APC cytophagy, or both immunne response of having concurrently.
Described vaccine is polysaccharide vitreous can further to be dispersed in functional macromolecular solution, is prepared into microgranule (0.5 μ m-10 μ m) or the face coat that is coated in various supports that sustained-release micro-spheres, immunocyte can engulf, is prepared into skeleton, suppository etc.
The vaccine and the antibody polysaccharide vitreous particle smooth surface rounding of the inventive method preparation, the granule regular without adhesion, particle diameter can be regulated and control from 0.1 μ m as required to 10 μ m.Further vaccine with antibody is polysaccharide vitreous is dispersed in the controlled release high molecular material, prepare different dosage forms,, can keep activity and therapeutic effect for a long time as minimicrosphere of sustained-release micro-spheres, the cytophagic 0.5 μ m-10 μ m of APC etc. according to clinical needs.The preparation be loaded with vaccine or the antibody polysaccharide vitreous particle can be directly used in inhalant dosage form, the protection fragile structure vaccine or antibody; The temperature-sensitive liquid-glue body that is loaded with vaccine or antibody polysaccharide vitreous particle can be directly used in clinical subcutaneous injection administration or implant the effect of subcutaneous slow controlled release vaccine or antibody.The vitreous body that is loaded with vaccine or antibody can be prepared into sustained-release micro-spheres by S/O/W or S/O/O emulsion method, particle diameter is at 500nm-10, the microsphere of 000nm can inducing cell immunity or cell and humoral immunization, and can delaying the controlled release vaccine at 20 μ m-120 μ m microspheres, to play with humoral immunization be main immunization therapy and prevention.Polysaccharide not only can be protected vaccine or antibody mutually in the preparation process, can also be used to improving its release dynamics.For the microsphere of given vaccine or antibody loading, the consumption of polysaccharide be one easily factor can be used to the rate of release and the release mode of vaccine or antibody.Increase the consumption of polysaccharide, then rate of release improves, and not exclusively the problem that discharges improves, but the prominent probability of releasing increases.The consumption that reduces polysaccharide then plays reverse effect.The consumption of carefully selecting polysaccharide can be able to reach in conjunction with the molecular weight of degradable macromolecule and hydrophilic-hydrophobic simultaneously both having controlled prominent releasing, and avoided the purpose that not exclusively discharges again.
The specific embodiment
The primary condition of embodiment: vaccine or antibody--the polysaccharide vitreous preparation and the preparation of microsphere or year protein-polysaccharide vitreous responsive type preparing gel.Can be according to the character of concrete vaccine and antibody, can select three kinds of methods that prepare vaccine or antibody-polysaccharide vitreous a kind of, two or three.
Embodiment 1
1. low temperature aqueous phase-aqueous phase emulsion legal system is equipped with vaccine or antibody-polysaccharide vitreous
1. prepare vaccine or antibody, PEG and polysaccharide solution
Prepare 10% PEG and 10% polysaccharide solution respectively with ultra-pure water, accurately taking by weighing 10 gram PEG and 10 gram polysaccharide water is placed on respectively in 100 milliliters the beaker and adds water 90 grams, then two beakers are placed on the magnetic agitation 30min of heating, the polysaccharide for the treatment of PEG and 10% all dissolving to take off cooling stand-by.Accurately take by weighing 800 milligrams of vaccines or antibody is dissolved in 7.2 ml waters stand-by with electronic balance.
Be equipped with vaccine or antibody, PEG and polysaccharide aqueous phase-aqueous phase emulsion
Under 0 ℃ of-4 ℃ of condition, the polysaccharide with 1., vaccine or antibody and PEG aqueous solution are respectively 1: 1: 5,1: 1: 10,1: 1: 20,1: 1: 40 at the abundant mixing of cillin bottle vortex 30s-60s according to volume ratio, form aqueous phase-aqueous phase emulsion; Or to take by weighing mass ratio according to polysaccharide and PEG be to be dissolved in water at cillin bottle elder generation mixing in 1: 1: 5,1: 1: 10,1: 1: 20,1: 1: 40 again, is to add at 1: 1 according to the weight ratio of polysaccharide and vaccine or antibody again, makes it to form aqueous phase-aqueous phase emulsion.
Freeze vaccine or antibody, PEG and polysaccharide aqueous phase-aqueous phase emulsion
To prepare vaccine or antibody, PEG and polysaccharide and form aqueous phase-aqueous phase emulsion, some are not assigned to the protein of polysaccharide phase, are redistributed in the polysaccharide decentralized photo freeze overnight in temperature-fall period.
4. completing steps sample 3. is in the vacuum freeze drier lyophilizing;
5. completing steps sample is 4. removed PEG continuous phase for three times with washed with dichloromethane to carry vaccine polysaccharide vitreous.
The polysaccharide vitreous particle diameter of vaccine that obtains or antibody obtains the smooth rounding of these vitreous body mostly at 300nm-5 μ m, and particle size distribution is even.The protection that the structure of vaccine or antibody is obtained has been avoided at dosage form preparation process inactivation.
2. be loaded with the polysaccharide vitreous microsphere preparation of vaccine or antibody
Take by weighing PVA and the NaCl of 1g and 5g, add ultra-pure water 94g, heat simultaneously, stir,, stop heating, continue slowly to stir to the PVA complete swelling, treat that solution is clear and does not have bubble after, stop to stir, it is stand-by to be put in 4 ℃ of refrigerators.This solution concentration is 1%PVA (W/W) and 5%NaCl (W/W).
Accurately take by weighing polylactic acid-polyglycolic acid (PLGA) 200mg, the 980mg that adds methylene chloride, vortex oscillation is dissolved in the dichloromethane PLGA, gets the dichloromethane solution of PLGA, and concentration is 20% (W/W), and matching while using is in case the dichloromethane volatilization.
Take by weighing vaccine or antibody polysaccharide vitreous particle 10mg, be dispersed in the dichloromethane solution of PLGA of 0.5g, the concentration of PLGA is 20% (W/W), magnetic agitation 15 minutes, rotating speed 1800rpm, fully stirring makes protein-polysaccharide vitreous particle be dispersed in the PLGA solution, the gained colostrum is scattered in the solution that contains 1%PVA and 10% sodium chloride once more, rotating speed homogenate with 2200rpm becomes emulsion (S/O/W), in one minute, transfer in 4 ℃ 10% sodium-chloride water solution, stir and collect aged protein microsphere after 3～4 hours, with ultra-pure water washing three times, spend the night ice phase pre-freeze, vacuum lyophilization again, making particle diameter is the microsphere of 50 μ m-120 μ m.The preparation microsphere can also can be used for subcutaneous injection with the microsphere of S/O/O multi-emulsion method and spray drying method for preparation, plays slow release and therapeutical effect.
Embodiment 2
1. the low temperature induction phase separation method prepares vaccine or antibody-polysaccharide vitreous
1. prepare vaccine or antibody, PEG and polysaccharide solution
Prepare 5% PEG and 5% polysaccharide solution respectively with ultra-pure water, accurately taking by weighing 5 gram PEG and 5 gram polysaccharide water is placed on respectively in 100 milliliters the beaker and adds water 95 grams, then two beakers are placed on the magnetic agitation 30min of heating, the polysaccharide for the treatment of PEG and 5% all dissolving to take off cooling stand-by.Accurately taking by weighing 400 milligrams of albumen with electronic balance is dissolved in 3.6 ml waters stand-by.
2. vaccine or antibody, PEG and polysaccharide homogeneous phase are total to aqueous solution
1. polysaccharide, vaccine or antibody and PEG aqueous solution are respectively 1: 1: 5,1: 1: 10,1: 1: 20,1: 1: 40 at the abundant mixing of cillin bottle vortex 30s-60s according to volume ratio, form homogeneous phase solution; Or to take by weighing mass ratio according to polysaccharide and PEG be to add water again at cillin bottle elder generation mixing in 1: 1: 5,1: 1: 10,1: 1: 20,1: 1: 40, makes it dissolving and form homogeneous phase solution.
3. freezing vaccine or antibody, PEG and polysaccharide homogeneous phase be aqueous solution altogether
To prepare the common aqueous solution of vaccine or antibody, PEG and polysaccharide homogeneous phase and be separated in temperature-fall period, and make polysaccharide become decentralized photo, PEG becomes continuous phase.But avoid freeze overnight under the quick-freezing condition.
4. completing steps sample 3. is in the vacuum freeze drier lyophilizing;
5. completing steps sample 4. removes with washed with dichloromethane that PEG continuous phase to be carried vaccine or antibody is polysaccharide vitreous for three times.
2. be loaded with the polysaccharide vitreous microsphere preparation of vaccine or antibody
Take by weighing PVA and the NaCl of 1g and 5g, add ultra-pure water 94g, heat simultaneously, stir,, stop heating, continue slowly to stir to the PVA complete swelling, treat that solution is clear and does not have bubble after, stop to stir, it is stand-by to be put in 4 ℃ of refrigerators.This solution concentration is 1%PVA (W/W) and 5%NaCl (W/W).
Accurately take by weighing polylactic acid-polyglycolic acid (PLGA) 200mg, the 980mg that adds methylene chloride, vortex oscillation is dissolved in the dichloromethane PLGA, gets the dichloromethane solution of PLGA, and concentration is 20% (W/W), and matching while using is in case the dichloromethane volatilization.
Take by weighing vaccine or antibody polysaccharide vitreous particle 10mg, be dispersed in the dichloromethane solution of PLGA of 0.5g, the concentration of PLGA is 20% (W/W), magnetic agitation 15 minutes, rotating speed 1800rpm, fully stirring makes protein-polysaccharide vitreous particle be dispersed in the PLGA solution, the gained colostrum is scattered in the solution that contains 1%PVA and 10% sodium chloride once more, rotating speed homogenate with 2200rpm becomes emulsion (S/O/W), in one minute, transfer in 4 ℃ 10% sodium-chloride water solution, stir and collect aged protein microsphere after 3～4 hours, with ultra-pure water washing three times, spend the night ice phase pre-freeze, vacuum lyophilization again, making particle diameter is the microsphere of 50 μ m-120 μ m.
The preparation microsphere can also can be used for subcutaneous injection with the microsphere of S/O/O multi-emulsion method and spray drying method for preparation, plays slow release and therapeutical effect.
Embodiment 3
Stable aqueous phase-aqueous phase emulsion method
Year vaccine that obtains or the Vitrea particle diameter great majority of antibody polysaccharide decentralized photo obtain the smooth rounding of these vitreous body all at 300nm-5 μ m, and particle size distribution is even.The protection that the structure of vaccine or antibody is obtained has been avoided at dosage form preparation process inactivation.
Take by weighing polylactic acid-polyglycolic acid (PLGA) 100mg, make it under the effect of eddy mixer, fully to be dissolved in the acetonitrile of 1ml, take by weighing then in the solution of the vaccine of 10mg or the polysaccharide vitreous adding acetonitrile of antibody, and under magnetic agitation, fully disperse.The surfactant span80 that outside a certain amount of, adds 200ml Oleum Gossypii semen in the oil-phase solution, abundant mixing under motor stirrer stirs with 200rpm.Dropwise add in the outer oil-phase solution in the stirring being dispersed with the polysaccharide vitreous acetonitrile solution of vaccine or antibody, stir and solidified 5 hours, sucking filtration, the microsphere particle that obtains is given a baby a bath on the third day after its birth inferior with the 50ml petroleum ether, drying under reduced pressure 10 hours, collect microsphere, the particle diameter of observing microsphere at existing micro mirror is the microsphere of 50 μ m～120 μ m, meets hypodermic requirement.
Can delay the controlled release vaccine, play and keep intravital vaccine concentration, thereby the humoral immunization of exciting is kept internal antibody concentration and reached treatment and prevention purpose.
Embodiment 4
Be loaded with the polysaccharide vitreous temperature sensitive type preparing gel of vaccine or antibody
Take by weighing vaccine or antibody polysaccharide vitreous particle 10mg, in the aqueous solution of 4 ℃ of PLGA-PEG-PLGA that are dispersed in 0.5g, the concentration of PLGA-PEG-PLGA is 20% (W/W), magnetic agitation 15 minutes, rotating speed 1800rpm, fully stirring makes vaccine or antibody polysaccharide vitreous particle be dispersed in the PLGA-PEG-PLGA solution, and the suspension that gained is dispersed in PLGA-PEG-PLGA is heated to 37 ℃ of curing, does release in vitro.Clinical practice is then now with the current.
Embodiment 5
The preparation and the release in vitro behavior of model vaccine BSA controlled release agent
Can under the temperate condition of the natural structure picture that keeps vaccine composition or antibody, be made into polysaccharide vitreous particle.This microgranule has the toleration to conditions such as organic solvent, temperature; can further the vaccine granule that is protected be made such as various titres of keeping intravital vaccine concentration, improving antibody such as high molecular slow-release microspheres, perhaps offer the irritation cell immunity by cross-reacting antigen.
One, solution allocation:
1.BSA formulations prepared from solutions: take by weighing Myoglobin 20.7mg, add choosing dissolving in whirlpool among the ultra-pure water 2ml, obtain the about 10mg/ml of Myoglobin solution concentration.
2. dextran solution preparation: take by weighing glucosan 1.0g, add ultra-pure water to 10.00g, heated and stirred is to dissolving.Obtain dextran solution percentage by weight about 10%.
3.PEG formulations prepared from solutions: take by weighing glucosan 1.0g, add ultra-pure water to 10.00g, heated and stirred is to dissolving.Obtain dextran solution percentage by weight about 10%.
Two, model vaccine BSA-dextran microparticles preparation:
1.BSA dextran microparticles preparation: take by weighing Myoglobin solution, dextran solution and PEG solution 0.50g, 0.16g and 1.00g (annotating two parts) respectively, under 0 ℃ condition, employing homogenizer E shelves (22,000rpm) homogenate 3 * 5s.Above-mentioned emulsion is let slip night at-20 ℃, again lyophilization 24h.Dry thing places dichloromethane 5ml, and 12, the centrifugal 5min of 000rpm inclines the upper strata stillness of night, repeats washed with dichloromethane process three times.The Myoglobin dextran microparticles that obtains at last is kept in the 1ml dichloromethane.
2. blank dextran microparticles preparation: take by weighing dextran solution and PEG solution 0.15g and 1.00g respectively, prepare with method by last noodle producing method.
(annotate: it is spherical in shape that above-mentioned two kinds of microgranules are measured its particle size distribution and size and scanning electron microscopic observation form at PCS, and the most of particle diameters of microgranule are about 1～2um.)
Three, the preparation of model vaccine BSA slow releasing preparation:
Film controlled release preparation preparation in 1: 5: take by weighing PLGA (75/253A) 15.1mg, dichloromethane 1ml, whirlpool choosing dissolving.Add the BSA dextran microparticles suspension that 700ul shakes up, vortex makes and is uniformly dispersed.Be prepared into the PLGA microsphere by the S/O/W method.We can be clearly seen that finely dispersed BSA dextran microparticles in the ball.
Four, discharging liquid measures:
Discharge the liquid sample and adopt the micro-BCA kit measurement.With BSA standard protein preparation standard curve.BSA-glucosan PLGA microsphere discharges the subduction of liquid absorption value to be proofreaied and correct with the blank microsphere release liquid absorption value of method preparation, brings into to calculate in the above-mentioned standard curve to discharge liquid BSA concentration.Draw the release profiles of BSA in different glucosans/PLGA ratio coating successively.Its release behavior is good as can be seen for we, does not have the obviously prominent behavior of releasing.
Embodiment 6
The preparation and the release in vitro behavior of model antibody β-half sugared lactoside pheron controlled release agent
Had 500 kinds of antibody to be used for diagnosis and treatment at present in the world, FDA has ratified 20 antibody listings, wherein 8 kinds of targeting antibodies that are used for oncotherapy.As antibody Avastin at vascular epidermis somatomedin [VEGF], make late period colon cancer patient on average prolong five months life cycle, but medical expense and costliness thereof.Because the molecular weight of antibody is bigger, complex structure, common quarternary structure, under the rugged environment, the structure picture is easier to change in vivo, than the easier inactivation of chemical medicine.Therefore need the system of one conveying complete structure antibody, directly deliver to around the blood vessel of tumor cell, suppress the angiogenic growth of tumor cell.Or the antibody of other killing tumor cell, play local sustained release antibody, improve local blood drug level, reduce the consumption and the toxicity of medicine, save the treatment cost.Still selected one with antibody molecule amount sizableness, also have the protective effect that the sugared lactoside pheron of the model protein β of space quarternary structure-half illustrates this confrontation between systems body.
One, the sugared lactoside pheron of model β-half-dextran microparticles preparation is according to example 5 preparations
Two, preparation of model vaccine BSA-dextran microparticles and microsphere preparation are according to example 5 preparations
Three, release test: discharge liquid according to example 5 and measure;
Four, the inventive method is to the protective effect of β-half sugared lactoside pheron
In order to verify that the present invention has the better protect effect to bioactive substance; design following experiment: dextran microparticles is carried dextran microparticles to the sugared lactoside enzyme of β-half (have quarternary structure, molecular weight is the enzyme of 500KD) and is tested the protective effect of the protein macromolecule of structure mutability under the organic solution existence condition.Earlier protein is dissolved in dextran solution with the sugared lactoside enzyme of 0.67mg/ml β-half,, then it is added emulsifying in the example 1 described PEG solution according to 1: 20 ratio.After the lyophilization, several all over removing PEG with the dichloromethane cyclic washing.In the following step, the proteinic dextran microparticles that carries that is reclaimed is dissolved in buffer again, add o-nitrophenyl-β-D-galactopyranoside (ONPG), test is to the hydrolyzation catalysis activity of substrate-ONPG.The catalytic action of enzyme has just descended less than 10% having experienced from example 5 to example after 6 the operation described processes (comprising emulsifying, lyophilization and washed with dichloromethane).This activity of 10% descends and has also comprised the protein active that loses owing in Partial Protein that is dispensed on the PEG phase in emulsion process and the freezing dry process.That is to say, with the contacting of the used organic solution-dichloromethane of microcapsule packet procedures in, dextran microparticles has been preserved protease activities effectively.
Embodiment 7
The particulate vaccine of being engulfed by antigen presenting cell (APC) prepares
When the particle diameter of microgranule at 500nm-10,000nm is easily by the APC cytophagy, some antigens can escape into cytoplasm from the endocytosis body by different approach.This approach claims phagosome-cytosol (phagosome-cytosol) mode again; Or the MHCI quasi-molecule directly enters endosome through endoplasmic reticulum and Golgi body and combines with exogenous antigen, and it is offered cell surface; Exogenous antigenic peptides combines with the MHCI quasi-molecule of cell surface outside exocytosis is released to born of the same parents in lysosome.Offer principle based on above-mentioned non-classical exogenous antigen MHCI quasi-molecule, designed the complete structure that guarantees vaccine, and use the macromolecular material (PEI) that can help vaccine to escape out from the endocytosis body, guaranteed more vaccine from the endocytosis body to cytoplasm, thereby the immunity of more opportunity irritation cell is arranged.
One, solution allocation:
1. hepatitis E vaccine solution preparation: take by weighing hepatitis E vaccine 4.05mg, add choosing dissolving in whirlpool among the ultra-pure water 1ml, obtain the about 4.05mg mg/ml of hepatitis E vaccine solution concentration.
2. dextran solution preparation: take by weighing glucosan 1.0g, add ultra-pure water to 10.00g, heated and stirred is to dissolving.Obtain dextran solution percentage by weight about 10%.
3.PEG formulations prepared from solutions: take by weighing glucosan 1.0g, add ultra-pure water to 10.00g, heated and stirred is to dissolving.Obtain dextran solution percentage by weight about 10%.
Two, model vaccine hepatitis E vaccine-glucosan nanoparticle preparation:
1. hepatitis E vaccine dextran microparticles preparation: take by weighing hepatitis E vaccine solution, dextran solution and PEG solution 0.05g, 0.02g and 8.00g (annotating two parts) respectively, another PEI that divide to add micromolecule 1mg at polysaccharide under 0 ℃ condition, employing homogenizer E shelves (22,000rpm) homogenate 3 * 5s.Above-mentioned emulsion is let slip night at-20 ℃, again lyophilization 24h.Dry thing places dichloromethane 5ml, and 12, the centrifugal 5min of 000rpm inclines the upper strata stillness of night, repeats washed with dichloromethane process three times.The hepatitis E vaccine dextran microparticles that obtains at last is kept in the 1ml dichloromethane.
2. blank dextran microparticles preparation: portion takes by weighing dextran solution and PEG solution 0.02g and 8.00g and another part 0.02g dextran solution and the PEI and PEG solution and the 8.00g that add 4mg respectively, prepares with method by last noodle producing method.
Three, the preparation of the PLGA microsphere of model vaccine hepatitis E vaccine-glucosan nanoparticle:
1. take by weighing vaccine hepatitis E vaccine-glucosan nanoparticle 10mg, 50mg PLGA50/50 (mean molecule quantity 6,500) be dissolved in the 1ml acetonitrile, the Oleum Gossypii semen 8ml that contains 0.5% (w/v) span, with two liquid mixing, 10krpm homogenate 30-60 second, microscopically is observed less than 10um (preparation of S/O/O method), this emulsion is splashed into 100ml Oleum Gossypii semen extraction volatilization 4 hours, add last filtration of 100ml petroleum ether 15min. at last and volatilize.
Four, the immunocompetent recovery of the preparation process of the PLGA microsphere of hepatitis E vaccine-glucosan nanoparticle
Take by weighing hepatitis E vaccine-glucosan nanoparticle and the 2mg PLGA microsphere of 2mg respectively, wherein microsphere 4ml washed with dichloromethane 4 times, centrifugal with 1200 commentaries on classics/min, remove supernatant.Volatilize at last and be dissolved in phosphate buffer again, the reagent that reuse Xiamen University provides closes (ELISIA) and measures.The immunocompetence of its recovery, we find to remove Physical Loss or Damage, activity almost all keeps.Can prevent hepatitis E.
Claims (5)
1, the preparation method of the polysaccharide vitreous particle of a kind of carrying active vaccine or antibody is characterized in that, concrete steps are as follows:
1. vaccine or antibody are added polysaccharide solution, described polysaccharide is a glucosan;
2. the polyglycol solution of prepared vaccine or antibody-polysaccharide 6% or 6% above concentration solution and same concentrations is mixed being lower than under the temperature that room temperature is higher than freezing point, or vaccine or antibody-polysaccharide 6% or 6% above concentration solution mixed in temperature above freezing with the polyglycol solution of the polyelectrolyte that contains 0.2-2%, or vaccine or antibody-polysaccharide 6% following concentration solution are mixed in temperature above freezing with the same concentrations polyglycol solution;
3. formed mixed solution or water-water milkiness liquid cooling lyophilizing is dry;
4. resulting lyophilized powder is added in the dichloromethane of soluble polyethylene glycol and insoluble polysaccharide and washs, remove Polyethylene Glycol, obtain being loaded with vaccine or antibody-polysaccharide vitreous particle;
Described Polyethylene Glycol, its molecular weight are 1,000-30,000; Its concentration is between the 0.2%-50%;
Described polyelectrolyte is any in sodium alginate, the sodium carboxymethyl cellulose.
2, the preparation method of the polysaccharide vitreous particle of carrying active vaccine according to claim 1 or antibody is characterized in that, described polysaccharide, its molecular weight are 10,000-2,000,000; Its concentration is 2%-20%.
3, the preparation method of the polysaccharide vitreous particle of carrying active vaccine according to claim 1 or antibody is characterized in that described polysaccharide dispersion particle, contains salt and micromolecule saccharide material.
4, the preparation method of the polysaccharide vitreous particle of carrying active vaccine according to claim 1 or antibody is characterized in that, described vaccine is subunit vaccine, live-virus vaccine, deactivation viral vaccine, dead viral vaccine, nucleic acid vaccine.
5, the preparation method of the polysaccharide vitreous particle of carrying active vaccine according to claim 4 or antibody, it is characterized in that described subunit vaccine is: hepatitis A vaccine, Hepatitis B virus vaccine, hepatitis C vaccine, hepatitis E vaccine, AIDS vaccine, anthrax vaccine, tumor vaccine, SARS vaccine, influenza vaccines, schistosomicide vaccine; Described live-virus vaccine is Measles Vaccine, poliomyelitis vaccine, pestilence vaccine, bacillus calmette-guerin vaccine.
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| US10357559B2 (en) | 2013-12-27 | 2019-07-23 | Emergent Product Development Gaithersburg Inc. | Temperature stable vaccine formulations |
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