CN102258786B - Porous microspheres for medicine carriers, preparation method and medicine loading method - Google Patents
Porous microspheres for medicine carriers, preparation method and medicine loading method Download PDFInfo
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Abstract
The invention relates to the field of preparation of porous microspheres, in particular to porous microspheres for medicine carriers, a preparation method and a medicine loading method. The method comprises the following steps of: 1) dissolving a degradable di-block amphiphilic polymer material in an organic solvent to form an oil phase O; 2) adding the oil phase O into a water phase W which contains a stabilizing agent to form O/W pre-emulsion; 3) allowing the O/W pre-emulsion obtained in the step 2 to pass through a microporous membrane to obtain O/W emulsion; and 4) removing the organic solvent from the emulsion obtained in the step 3, and curing, performing centrifugal washing and freeze drying to obtain porous medicine carrying microspheres. The organic solvent in the step 1) at least comprises a solvent of which the solubility in water is less than 2 percent; and the volume ratio of the oil phase O to the water phase W is 1:5-20. The porous microspheres overcome the problem that the conventional sustained release microspheres cannot be completely released at a later stage, and can effectively keep the medicine activity in the processes of carrying, storing and releasing the medicines.
Description
Technical field
The present invention relates to the preparation field of porous microsphere, particularly, the present invention relates to a kind of porous microsphere for pharmaceutical carrier, preparation method and drug loading method.
Background technology
Along with the development of emerging biometric technology and genetic engineering, the biochemical medicine of proteins and peptides class macromole (such as growth hormone, insulin, heparin, interferon, interleukin etc.) because show in dwarfism, diabetes, liver cirrhosis and cancer etc. are difficult to the therapeutic process of cure diseases that pharmacological action is strong, few side effects and the characteristics that seldom cause allergic reaction and extremely pay attention to.Because the Peptides and proteins poor stability easily is degraded in the intestines and stomach, and has the low problem of bioavailability, therefore adopt injection more.But because the Half-life in vivo of this type of medicine is short, usually need the multiple injection administration during clinical practice.For these problems, researcheres are followed two kinds of solution routes substantially, and the firstth, the route of administration of a high bioavailability of pharmaceutical grade protein of searching; The secondth, optimize the dynamic metabolism characteristic of protein by chemical modification, to prolong its half-life, reach long-acting stable purpose.Wherein, utilize biology can fall the type polymer and prepare Loaded Microspheres Drug Delivery System for framework material, can reach long-acting slow-release, reduce administration number of times and medicine irritation, reduce toxic and side effects, improve the purpose of curative effect.Usually embedding medicinal adopts multi-emulsion method, and the contacting of albumen and organic solvent and oil-water interfaces in preparation process all may affect the structure of protein; And stir, the interference of these extraneous factors of supersound process easily makes series of physical or the chemical changes such as it is assembled, absorption, precipitation, oxidation, deacylated tRNA amine, hydrolysis.In a single day protein structure is destroyed, and not only drug effect descends, and may produce in vivo immunogenicity and other untoward reaction.In order to increase the stability of protein drug in the microsphere process; generally need to its with carry out frozen dried after some protective agent solution mix; protective agent commonly used mainly contains two classes: polyethylene glycols and saccharide; but residual mensuration and removal are brought difficulty, be unfavorable for large-scale industrial production.
In order to overcome easily destroyed problem of protein and other pharmaceutically actives in the embedding process, can reach the medicine carrying purpose with the microsphere absorption medicine that loose structure is arranged.Because have the conventional microsphere that the microsphere of loose structure is compared equal size, more specific surface area can be provided, when preparing microsphere agents of drug carriers with absorption method, can significantly improve the useful load of medicine.The homogeneity of particle diameter is the key issue that microsphere is used, because the particle diameter heterogeneity certainly will cause the problem of preparation repeatability and treatment poor repeatability, is difficult to declaration clinical.Use the porous polylactic acid microball radius particle pore distribution heterogeneity of paddling process preparation in the Chinese patent (publication number CN101361716A), be difficult to improve carrying drug ratio and accurately regulating medicine discharge and experiment batch between repeatability.
Can discharging fully of drug loading also is the subject matter that restriction drug-carrying polymer microsphere enters clinical practice, non-specific adsorption easily occurs between hydrophobic polymeric material and the protein drug that loads, the incomplete release that causes medicine causes the waste of medicine and bioavailability to reduce.The micro-sphere material of the absorption medicine that uses in the Chinese patent (Granted publication CN100388970C) is hydrophobic polylactic acid (PLA) copolymer, this hydrophobic material easily causes the incomplete release of medicine in the later stage of protein medicaments discharges.And the non-specific adsorption effect between film material and the albumen easily makes albumen assemble, and reduces the activity of medicine.
For different diseases, required drug release rate is different, for example, is used for the treatment of anticancer medicine needs and discharges in a large number timely, to reach effective drug level; And be used for the treatment of the medicines such as insulin of diabetes, and need slowly to discharge guarantee stable drug level, reduce frequency injection and alleviate patient's misery.Therefore, the controlled medicine carrying microballoons of preparation rate of release is very necessary.Uses ethyl cellulose to prepare the controlled porous microsphere of particle diameter as the film material in the Chinese patent (publication number CN1939281A), but the degradation speed of microsphere is not regulated and control and studied, be unfavorable for the universality of drug use.Generally speaking, when preparation porous medicine carrying microballoons, need to add perforating agent, cause residual mensuration difficulty of later stage, and reduce biological safety; And the microspherulite diameter that existing technology prepares and aperture heterogeneity, carrying drug ratio is low, poor repeatability; In the dispose procedure protein drug easily with microsphere skeleton generation non-specific adsorption, causes the medicine incomplete release and produce aggregation, thereby the generation immunogenicity causes losing activity; The microsphere degradation process is uncontrollable, is unfavorable for that expanded application is in the treatment of various diseases.
The present invention is directed to porous microsphere radius particle pore distribution heterogeneity, carrying drug ratio is low, can not control and discharge and the problem such as pharmaceutically active is low, and proposing to adopt a kind of amphipathic nature polyalcohol of two blocks is material, and the preparation porous microsphere solves an above-mentioned difficult problem as pharmaceutical carrier.
Summary of the invention
The object of the present invention is to provide a kind of porous microsphere for pharmaceutical carrier.
A further object of the present invention is to provide a kind of preparation method of the porous microsphere for pharmaceutical carrier.
An also purpose of the present invention is to provide a kind of drug loading method of the porous microsphere for pharmaceutical carrier.
According to the porous microsphere for pharmaceutical carrier of the present invention, the preparation method of described microsphere may further comprise the steps:
1) degradable two block amphipathic nature polyalcohol materials is dissolved in the organic solvent, forms oil phase O;
2) oil phase O is joined the aqueous phase W that contains stabilizing agent, form the pre-breast of O/W;
3) with step 2) the pre-breast of the O/W of gained is by microporous membrane, obtains the O/W emulsion;
4) with step 3) organic solvent in the resulting emulsion removes, solidifies, and again through centrifuge washing and lyophilization, obtains the porous medicine carrying microballoons;
Described step 1) in organic solvent contain at least a kind of in water dissolubility be lower than 2% solvent, described organic solvent comprises one or more in dichloromethane, chloroform, Carbon bisulfide and the dimethylbenzene;
Described step 2) volume ratio 1: 5～20 of oil phase O and water W in.
According to the porous microsphere for pharmaceutical carrier of the present invention, the size of described pre-emulsion is more preferably greater than membrane aperture, the preparation method of pre-emulsion, can be by method preparations such as common emulsifying manner such as homogenizing, ultrasonic, mechanical agitation, the removal of the organic solvent in the described emulsion can adopt reduction vaporization, normal temperature and pressure to stir in the methods such as volatilization or solvent extraction one or several.
According to the porous microsphere for pharmaceutical carrier of the present invention, described pressure can be regulated between 1-2000kPa, this mainly by the preparation of the size in the microporous membrane aperture of using in the preparation process and targeted microspheres size require determine.
According to the porous microsphere for pharmaceutical carrier of the present invention, described microporous membrane comprises hydrophobic and hydrophilic film, preferred hydrophilic film, as adopting hydrophilic SPG film, this SPG film is commercial prod, in preparation process, can control by the SPG film of selecting different membrane aperture the size of product, microporous membrane aperture commonly used is 0.5-200 μ m, and preferably 1.4-50 μ m is more preferably 2.8-18 μ m.
According to the porous microsphere for pharmaceutical carrier of the present invention, the selection face of described polymeric material is very wide, not only can select amphipathic nature material, as polylactic acid, PLGA, polycaprolactone, poe, poly-anhydride, polyphosphazene respectively with the polymeric material of Polyethylene Glycol copolymerization gained in any one, also the composite mixing of the polymer of its variety classes different molecular weight can be used.
According to the porous microsphere for pharmaceutical carrier of the present invention, described organic solvent can be selected from the organic solvent that dissolubility in the water is lower than 2% (not comprising 2%), as in the organic solvents such as dichloromethane, chloroform, Carbon bisulfide and dimethylbenzene any one or multiple, concrete kind or volume need be decided on preparation parameters such as used film materials.
According to the porous microsphere for pharmaceutical carrier of the present invention, described step 3) can be repeatedly, be about to step 3) emulsion of gained as pre-breast with pressure again by microporous membrane, until the size of the emulsion that obtains and homogeneity meet the demands.
According to the porous microsphere for pharmaceutical carrier of the present invention, described outer water stabilizing agent optional from polyvinyl alcohol, polyglyceryl fatty acid ester, Tween-81 (Tween80), polyoxyethylene sorbitol acid anhydride laurate (Tween20), dodecyl sodium sulfate (SDS) etc. one or more, the stabilizing agent working concentration is preferably 0.1%～10wt%.
The present invention also provides and has adopted described porous microsphere to carry out the method for drug loading, and the method may further comprise the steps:
By 1: 2～100 mass ratio medicine is mixed with porous microsphere, vibrating makes porous microsphere to Drug absorbability, centrifugal collection microsphere, and the medicine that flush away does not adsorb, lyophilization obtains adsorbing the porous of medicine medicine carrying microballoons is arranged;
Described medicine is to be selected from a kind of in polypeptide, protide, polysaccharide, nucleic acid, the vaccine, the medicine carrying microballoons that the present invention prepares has very high release rate and keeps good biological activity for medicine, especially more obvious for polypeptide, protein matter effect, well solved an above-mentioned difficult problem.Because structure and polypeptide when polysaccharide, nucleic acid and vaccine medicine contact under the strong preparation condition such as ultrasonic, stirring and with organic solvent, protein matter are the same, very easily destroy and cause inactivation, therefore, by porous microsphere this class medicine is adsorbed the preparation medicine carrying microballoons in the present invention, can well keep pharmaceutically active, have equally good effect.
The present invention also provides a kind of preparation method of the porous microsphere for pharmaceutical carrier, said method comprising the steps of:
1) degradable two block amphipathic nature polyalcohol materials is dissolved in the solvent, forms oil phase O;
2) oil phase O is joined the aqueous phase W that contains stabilizing agent, form the pre-breast of O/W;
3) with step 2) the pre-breast of the O/W of gained is by microporous membrane, obtains the O/W emulsion;
4) with step 3) organic solvent in the resulting emulsion removes, solidifies, and again through centrifuge washing and lyophilization, obtains the porous medicine carrying microballoons;
Described step 1) in solvent contain at least a kind of in water dissolubility be lower than 2% organic solvent, described organic solvent comprises one or more in dichloromethane, chloroform, Carbon bisulfide and the dimethylbenzene;
Described step 2) volume ratio 1: 5～20 of oil phase O and water W in.
The porous microsphere of size homogeneous provided by the invention, this microsphere size homogeneous, controlled, diameter Distribution coefficient (CV) are worth in 20%, and be preferred in 15%.The medicine charging ratio reaches as high as 50%, and protein and peptide drugs activity keeping rate can abrupt release or 1 thoughtful 12 weeks of sustained release more than 90% in the dispose procedure.Particle size distribution coefficient (Coefficient of Variation, CV) adopts number ball method to measure, and measures the particle diameter of 300 particles, calculates the particle size distribution coefficient (CV) of particle by following formula.
D wherein
iBe the particle diameter of single particle,
Be the number average bead diameter of particle, N is the sum of particle, N＞300.
According to the porous microsphere for pharmaceutical carrier of the present invention, the average particle size range of described microsphere is at 500nm-100 μ m, described polymer microballoon has adopted at least a kind of amphipathic nature polyalcohol material of two blocks and has adopted at least a kind of dissolubility in water to be lower than the organic solvent of 2% (not comprising 2%) in preparation process, more be conducive to improve medicine charging ratio, keep biological activity and sustainable release.
Adopt the fast film emulsification method to prepare particle diameter among the present invention and the aperture homogeneous is controlled, carrying drug ratio is high, can control release, and the much higher hole of pharmaceutically active microsphere.Wherein, the film emulsifying technology has guaranteed that the medicine carrying microballoons uniform particle diameter is controlled, the method preparations such as common emulsifying manner such as homogenizing, ultrasonic, mechanical agitation are at first selected in preparation during emulsion, the emulsion particle diameter that makes by these conventional methods is greater than membrane aperture, then under the effect of pressure with these particle diameters greater than the pre-emulsion of membrane aperture by behind the microporous membrane, just can obtain the particle diameter double emulsion consistent with microporous membrane, can repeated operation until the size of the emulsion that obtains and homogeneity meet the demands.
Secondly, the di-block copolymer of employing contains hydrophilic segment, in solidification process, because organic solvent produces quick bumping and pore-forming at microsphere surface, because hydrophilic segment distributes at the microsphere surface homogeneous, so bumping speed is identical, aperture, the duct homogeneous of generation; Simultaneously, by film material and the control oil-water ratio that adopts different hydrophilic segment proportion, can control the aperture of microsphere.
Again, because the specific surface area of porous microsphere obviously improves with respect to smooth microsphere, thereby the increase of the site of absorption, can obtain higher carrying drug ratio.Can control the degradation rate of microsphere by the molecular weight of controlling diaphragm material and the concentration in organic solvent, hydrophilic block proportion, oil-water ratio and the concentration of polymer in organic solvent all can affect the degradation rate of microsphere in film material molecular weight, the polymer, thereby can control by regulating above parameter the rate of release of medicine.Polymer molecular weight is lower, and the concentration of polymer in organic solvent is lower, and the microsphere skeleton is more loose, be beneficial to the degraded of microsphere, thereby drug release rate is very fast; Otherwise, improve polymer molecular weight and the concentration of polymer in organic solvent, microsphere skeleton consolidation, the microsphere degraded is slow, and rate of releasing drug is also slow.Wherein the shared ratio of hydrophilic block is higher in the polymer, and the chance of microsphere and ambient water environmental exposure is more, and the speed of hydrophilic chain degraded is faster, and the microsphere aperture is also along with the ratio of hydrophilic block increases and increases, thereby improves drug release rate; Otherwise the shared ratio of hydrophilic block wherein in the reduction polymer, rate of releasing drug decreases.Outer water volume is larger, and the microsphere aperture is larger, and dashing forward, it is larger to release; Otherwise outer water volume reduces, and the aperture is less, dashes forward and releases reduction, therefore can prepare different porous medicine carrying microballoonss according to state of an illness needs, thereby reaches the purpose of control drug release rate; Simultaneously, hydrophilic segment is distributed in microsphere surface, has avoided the non-specific adsorption of hydrophobicity microsphere and albumen and the incomplete release that causes.Absorption method prepares medicine carrying microballoons and has avoided the preparation conditions such as ultrasonic, stirring and organic solvent and oil-water interfaces on the impact of pharmaceutically active, well keeps pharmaceutically active.
The condition that is beneficial to the preparation porous microsphere among the present invention is: use dissolubility in water is lower than 2% organic solvent, and oil phase compares less than 1: 5 with water, and the hydrophilic block proportion is 4～20% in the amphipathic condensation material.If the organic solvent dissolubility in water that uses is higher than 2%, oil-water ratio was greater than 1: 5, and the hydrophilic block proportion is lower than 4% in the amphipathic condensation material, is unfavorable for preparing porous microsphere.If oil-water ratio was less than 1: 20, the hydrophilic block proportion is higher than 20% in the amphipathic condensation material, is unfavorable for preparing the microsphere of regular spherical.(the linear copolymer that is formed by hydrophobic segment and hydrophilic segment alternating polymerization because the amphipathic nature polyalcohol of two blocks, this polymer possesses hydrophilic and hydrophobicity simultaneously), can well solve the non-specific adsorption between protein medicaments and the microsphere film material, guarantee the fully release of medicine; In addition, can also stablize the microsphere of preparation aperture homogeneous take amphiphilic polymers as micro-sphere material, thereby can obtain higher carrying drug ratio.Because the hydrophilic parts itself in the amphipathic nature material has the character that is similar to perforating agent, does not need additionally to add perforating agent in preparation process, has reduced production cost, has solved the difficulty of residual mensuration, is beneficial to the raising biological safety.Adopt the fast film emulsion process can realize preparing uniform particle diameter, the porous microsphere that size is controlled because size and drug release are closely connected, thereby can reach the control purpose of deenergized period by regulating the microspherulite diameter size.
Preparation method disclosed by the invention compared with prior art has the following advantages:
The inventive method preparation efficiency is very high, and the flow velocity size when emulsion is crossed film is up to 10mls
-1, thereby preparation process most moment finish.
The invention provides a kind of porous medicine carrying microballoons, it is characterized in that, the diameter Distribution coefficient of described microsphere is in 20%, and the drug delivery rate is up to 50%, protein and peptide drugs activity keeping rate can abrupt release or 1 thoughtful 12 weeks of sustained release more than 90% in the dispose procedure.
The invention provides a kind of method of porous medicine carrying microballoons of quick preparation size homogeneous, and can be by microporous membrane pore size and the operating pressure size of controlling product of control in the preparation process.
The present invention has overcome the problem that prior art can't prepare the porous medicine carrying microballoons of uniform particle diameter, has guaranteed the repeatability of experiment, is beneficial to stability and the industrial amplification production of curative effect of medication.
The present invention need to additionally not add perforating agent at oil phase, can reach the effect of preparation porous microsphere, has removed the later stage from and has surveyed the residual difficulty of bringing, and is beneficial to simultaneously the reduction cost.
The present invention has overcome the problem that traditional slow release porous microsphere can not discharge fully in the later stage, and in drug loading, can both effectively keep pharmaceutically active in release and the storage process.
The inventive method is simple to operate, mild condition and be easy to industrial amplification production.
Description of drawings
Fig. 1 is the preparation flow schematic diagram of medicine carrying microballoons of the present invention;
Fig. 2 is the Electronic Speculum figure of the microsphere of embodiment 1 preparation;
Fig. 3 is the particle size distribution figure of the microsphere of embodiment 1 preparation;
Fig. 4 is the Electronic Speculum figure of the microsphere of embodiment 2 preparations;
Fig. 5 is the Electronic Speculum figure of the microsphere of embodiment 3 preparations;
Fig. 6 is the Electronic Speculum figure of the microsphere of embodiment 4 preparations;
Fig. 7 is the Electronic Speculum figure of the microsphere of embodiment 5 preparations.
The specific embodiment
The invention will be further described below in conjunction with embodiment, but the present invention is not limited among this embodiment.
Be that the hydrophilic porous film of 2.8 μ m places water to infiltrate with the aperture, make pore membrane fully moistening.Be that 10,000 PLA-PEG copolymer (PELA) (the shared ratio of hydrophilic block is 4%) is dissolved in the 8ml dichloromethane with the molecular weight of 0.4g, as oil phase, this oil phase is joined in Polyethylene Glycol (PVA) aqueous solution of the 1%wt of 80ml, the ratio of oil phase and water is 1: 10, magnetic agitation 300rpm stirs the pre-breast of 1min preparation, again this pre-emulsion was pressed down microporous membrane device (such as Fig. 1) at the operating pressure of 400kPa, obtain emulsion, emulsion is spent the film time less than 10s, again emulsion is at room temperature stirred 24h to remove organic solvent dichloromethane, namely obtain medicine carrying microballoons through centrifuge washing again.The microsphere vacuum drying 48h of gained is obtained the finished product microsphere.Dried microsphere is dispersed in water again, utilizes field emission scanning electron microscope (JEOL SEM Company, Japan) to observe the surface topography (such as Fig. 2) of microsphere.The volume average particle size of microsphere and distribution thereof are measured (such as Fig. 3) with laser particle analyzer (Malvern Company, USA), and the mean diameter of microsphere is 627nm after measured, and particle size distribution coefficient CV value is 12.53%.The 0.2g microsphere is placed little centrifuge tube, add 5mL glucagon-like-peptide-1 (GLP-1) solution, adsorb at a certain temperature 24h, GLP-1 solution centrifugalize under 5000rpm of then will not adsorb, measure the not GLP-1 quality of absorption, obtaining microsphere by calculation in quantity is 37.8% for the carrying drug ratio of GPL-1.Accurate weighing 30mg lyophilizing microsphere, PBS buffer (8g NaCl, 0.2g KCl, the 0.24g KH of adding 10ml pH7.4
2PO
4, 1.81gNa
2HPO
4H
2O, 0.5g NaN
3, 0.1g Tween20 and 1000ml distilled water).Sample cell places 37 ℃ of water-bath constant temperature oscillator joltings (120rpm).The 1.0ml supernatant is taken out in regularly centrifugalize, fills into simultaneously the fresh PBS buffer of 1.0ml.Protein content is then fixed with BCA reagent and micro-BCA test kit in the supernatant.After measured, microsphere sustained release GLP-1 accumulation reaches 97.2% in 6 weeks, and the GLP-1 activity keeping rate that discharges is 96.5%.
Embodiment 2
Be that the hydrophilic film of 7.2 μ m places water to infiltrate with the aperture, make pore membrane fully moistening.Be that 50,000 polylactic acid-polyglycolic acid and ethylene glycol copolymer (the shared ratio of hydrophilic block is 10%) are dissolved in the 10ml chloroform with the molecular weight of 0.7g, as oil phase, this oil phase is joined in the PVA aqueous solution of 1.2%wt of 50ml, the ratio of oil phase and water is 1: 5, magnetic agitation 300rpm stirs the pre-breast of 1min preparation, again this pre-emulsion was pressed down the microporous membrane device at the operating pressure of 200kPa, obtain emulsion, emulsion is spent the film time less than 10s, again emulsion is at room temperature stirred 24h to remove the organic solvent chloroform, namely obtain medicine carrying microballoons through centrifuge washing again.The microsphere vacuum drying 48h of gained is obtained the finished product microsphere.Dried microsphere is dispersed in water again, utilizes field emission scanning electron microscope (JEOL SEM Company, Japan) to observe the surface topography (such as Fig. 4) of microsphere.The volume average particle size of microsphere and distribution thereof are measured with laser particle analyzer (Malvern Company, USA), and after measured, the mean diameter of microsphere is 4.12 μ m, and particle size distribution coefficient CV value is 12.67%.The 0.2g microsphere is placed little centrifuge tube, add the 5mL insulin solutions, adsorb at a certain temperature 46h, the insulin solutions that then will not adsorb centrifugalize under 5000rpm, measure the not insulin quality of absorption, obtaining microsphere by calculation in quantity is 36.9% for the carrying drug ratio of insulin.Accurate weighing 30mg lyophilizing microsphere, PBS buffer (8g NaCl, 0.2gKCl, the 0.24g KH of adding 10ml pH7.4
2PO
4, 1.81g Na
2HPO
4H
2O, 0.5g NaN
3, 0.1g Tween20 and 1000ml distilled water).Sample cell places 37 ℃ of water-bath constant temperature oscillator joltings (120rpm).The 1.0ml supernatant is taken out in regularly centrifugalize, fills into simultaneously the fresh PBS buffer of 1.0ml.Protein content is then fixed with BCA reagent and micro-BCA test kit in the supernatant.After measured, the accumulation of microsphere sustained release insulin reaches 95.8% in 5 weeks, and the insulin active conservation rate that discharges is 94.7%.
Embodiment 3
Be that the hydrophilic film of 9 μ m places water to infiltrate with the aperture, make pore membrane fully moistening.Be that 20,000 PLA-PEG copolymer (the shared ratio of hydrophilic block is 20%) is dissolved in the 15ml Carbon bisulfide with the molecular weight of 0.8g, as oil phase, this oil phase is joined in the PVA aqueous solution of 1.4%wt of 300ml, the ratio of oil phase and water is 1: 20, magnetic agitation 300rpm stirs the pre-breast of 1min preparation, again this pre-emulsion was pressed down the microporous membrane device at the operating pressure of 150kPa, obtain emulsion, emulsion is spent the film time less than 10s, again emulsion is at room temperature stirred 24h to remove the organic solvent chloroform, namely obtain medicine carrying microballoons through centrifuge washing again.The microsphere vacuum drying 48h of gained is obtained the finished product microsphere.Dried microsphere is dispersed in water again, utilizes field emission scanning electron microscope (JEOL SEM Company, Japan) to observe the surface topography (such as Fig. 5) of microsphere.The volume average particle size of microsphere and distribution thereof are measured with laser particle analyzer (Malvern Company, USA), and after measured, the mean diameter of microsphere is 4.17 μ m, and particle size distribution coefficient CV value is 13.74%.The 0.2g microsphere is placed little centrifuge tube, add 5mL recombinant human growth hormone solution, adsorb at a certain temperature 36h, recombinant human somatropin's solution centrifugalize under 5000rpm of then will not adsorb, measure not recombinant human somatropin's quality of absorption, obtaining microsphere by calculation in quantity is 22.8% for recombinant human somatropin's carrying drug ratio.Accurate weighing 30mg lyophilizing microsphere, PBS buffer (8g NaCl, 0.2g KCl, the 0.24g KH of adding 10ml pH7.4
2PO
4, 1.81g Na
2HPO
4H
2O, 0.5g NaN
3, 0.1g Tween20 and 1000ml distilled water).Sample cell places 37 ℃ of water-bath constant temperature oscillator joltings (120rpm).The 1.0ml supernatant is taken out in regularly centrifugalize, fills into simultaneously the fresh PBS buffer of 1.0ml.Protein content is then fixed with BCA reagent and micro-BCA test kit in the supernatant.After measured, the accumulation of microsphere sustained release growth hormone reaches 94.8% in 7 weeks, and the growth hormone activity keeping rate that discharges is 95.4%.
Embodiment 4
Be that the hydrophilic film of 18 μ m places water to infiltrate with the aperture, make pore membrane fully moistening.Be that 30,000 polycaprolactone-polyethylene glycol copolymer (the shared ratio of hydrophilic block is 15%) is dissolved in the 20ml dimethylbenzene with the molecular weight of 1g, as oil phase, this oil phase is joined in the PVA aqueous solution of 1.5%wt of 160ml, the ratio of oil phase and water is 1: 8, magnetic agitation 300rpm stirs the pre-breast of 1min preparation, again this pre-emulsion was pressed down the microporous membrane device at the operating pressure of 100kPa, obtain emulsion, emulsion is spent the film time less than 10s, again emulsion is at room temperature stirred 24h to remove the organic solvent chloroform, namely obtain medicine carrying microballoons through centrifuge washing again.The microsphere vacuum drying 48h of gained is obtained the finished product microsphere.Dried microsphere is dispersed in water again, utilizes field emission scanning electron microscope (JEOL SEM Company, Japan) to observe the surface topography (such as Fig. 6) of microsphere.The volume average particle size of microsphere and distribution thereof are measured with laser particle analyzer (Malvern Company, USA), and after measured, the mean diameter of microsphere is 7.62 μ m, and particle size distribution coefficient CV value is 14.65%.The 0.2g microsphere is placed little centrifuge tube, add 5mL leuprorelin solution, adsorb at a certain temperature 24h, leuprorelin solution centrifugalize under 5000rpm of then will not adsorb, measure the not leuprorelin quality of absorption, obtaining microsphere by calculation in quantity is 47.8% for the carrying drug ratio of leuprorelin.Accurate weighing 30mg lyophilizing microsphere, PBS buffer (8g NaCl, 0.2g KCl, the 0.24gKH of adding 10ml pH7.4
2PO
4, 1.81g Na
2HPO
4H
2O, 0.5g NaN
3, 0.1g Tween20 and 1000ml distilled water).Sample cell places 37 ℃ of water-bath constant temperature oscillator joltings (120rpm).The 1.0ml supernatant is taken out in regularly centrifugalize, fills into simultaneously the fresh PBS buffer of 1.0ml.Leuprorelin content is then fixed with liquid phase in the supernatant.After measured, the accumulation of microsphere sustained release leuprorelin reaches 98.2% in 8 weeks, and the leuprorelin activity keeping rate that discharges is 97.9%.
Be that the hydrophilic film of 50.2 μ m places water to infiltrate with the aperture, make pore membrane fully moistening.Be that 40,000 PLA-PEG copolymer (the shared ratio of hydrophilic block is 6%) is dissolved in the composite solvent (volume is 1: 1) of 30ml dichloromethane and chloroform as oil phase with the molecular weight of 1.2g, this oil phase is joined in the PVA aqueous solution of 2wt% of 300ml, the ratio of oil phase and water is 1: 10, magnetic agitation 300rpm stirs the pre-breast of 1min preparation, again this pre-emulsion was pressed down the microporous membrane device at the operating pressure of 70kPa, obtain emulsion, emulsion is spent the film time less than 10s, again emulsion is at room temperature stirred 24h to remove organic solvent dichloromethane, namely obtain medicine carrying microballoons through centrifuge washing again.The microsphere vacuum drying 48h of gained is obtained the finished product microsphere.Dried microsphere is dispersed in water again, utilizes field emission scanning electron microscope (JEOL SEM Company, Japan) to observe the surface topography (such as Fig. 7) of microsphere.The volume average particle size of microsphere and distribution thereof are measured with laser particle analyzer (Malvern Company, USA), and after measured, the mean diameter of microsphere is 20.72 μ m, and particle size distribution coefficient CV value is 14.16%.The 0.2g microsphere is placed little centrifuge tube, add 5mL Hepatitis B virus vaccine solution, ultrasonic absorption 20h at a certain temperature, then the centrifugalize under 5000rpm of the metakentrin that will not adsorb (LHRH) solution, measure the not LHRH quality of absorption, obtaining microsphere by calculation in quantity is 39.2% for the carrying drug ratio of LHRH.Accurate weighing 30mg lyophilizing microsphere, PBS buffer (8g NaCl, 0.2g KCl, the 0.24g KH of adding 10ml pH7.4
2PO
4, 1.81g Na
2HPO
4H
2O, 0.5g NaN
3, 0.1g Tween20 and 1000ml distilled water).Sample cell places 37 ℃ of water-bath constant temperature oscillator joltings (120rpm).The 1.0ml supernatant is taken out in regularly centrifugalize, fills into simultaneously the fresh PBS buffer of 1.0ml.LHRH is then fixed with BCA reagent and micro-BCA test kit in the supernatant.After measured, microsphere sustained release LHRH accumulation reaches 99.0% in 4 weeks, and the LHRH activity keeping rate that discharges is 96.3%.
Claims (8)
1. a porous microsphere that is used for pharmaceutical carrier is characterized in that, the preparation method of described microsphere may further comprise the steps:
1) degradable two block amphipathic nature polyalcohol materials is dissolved in the organic solvent, forms oil phase O;
2) oil phase O is joined the aqueous phase W that contains stabilizing agent, form the pre-breast of O/W;
3) with step 2) the pre-breast of the O/W of gained is by microporous membrane, obtains the O/W emulsion;
4) organic solvent in the resulting emulsion of step 3) is removed, solidified, again through centrifuge washing and lyophilization, obtain the porous medicine carrying microballoons;
In the described step 1) organic solvent contain at least a kind of in water dissolubility be lower than 2% solvent;
Described step 2) the volume ratio 1:5 of oil phase O and water W～20 in;
Described step 1) two block amphipathic nature polyalcohol materials comprise the amphipathic condensation material of one or more and Polyethylene Glycol copolymerization gained in polylactic acid, PLGA, polycaprolactone, poe, poly-anhydride, the polyphosphazene, its molecular weight is 10,000～100,000, and the hydrophilic block proportion is 4～20% in this amphipathic condensation material;
Described step 2) stabilizing agent of aqueous phase comprises one or more in polyvinyl alcohol, polyglyceryl fatty acid ester, Tween-81, polyoxyethylene sorbitol acid anhydride laurate and the dodecyl sodium sulfate, and stabilizer concentration is 0.1wt%～10wt%;
The aperture of microporous membrane is 0.5～200 μ m in the described step 3), and the pressure by microporous membrane is 1～2000kPa.
2. the porous microsphere for pharmaceutical carrier according to claim 1 is characterized in that,
In the described step 1), described organic solvent comprises one or more in dichloromethane, chloroform, Carbon bisulfide and the dimethylbenzene.
3. the porous microsphere for pharmaceutical carrier according to claim 2, it is characterized in that, described organic solvent comprises one or both in dichloromethane and the chloroform, and described two block amphipathic nature polyalcohol materials comprise the amphipathic condensation material of polylactic acid, PLGA or polycaprolactone and Polyethylene Glycol copolymerization gained.
4. the porous microsphere for pharmaceutical carrier according to claim 1 is characterized in that, described medicine is to be selected from a kind of in polypeptide, the protide.
5. a preparation method that is used for the porous microsphere of pharmaceutical carrier is characterized in that, said method comprising the steps of:
1) degradable two block amphipathic nature polyalcohol materials is dissolved in the organic solvent, forms oil phase O;
2) oil phase O is joined the aqueous phase W that contains stabilizing agent, form the pre-breast of O/W;
3) with step 2) the pre-breast of the O/W of gained is by microporous membrane, obtains the O/W emulsion;
4) organic solvent in the resulting emulsion of step 3) is removed, solidified, again through centrifuge washing and lyophilization, obtain the porous medicine carrying microballoons;
In the described step 1) organic solvent contain at least a kind of in water dissolubility be lower than 2% solvent;
Described step 2) the volume ratio 1:5 of oil phase O and water W～20 in;
Described step 1) two block amphipathic nature polyalcohol materials comprise the amphipathic condensation material of one or more and Polyethylene Glycol copolymerization gained in polylactic acid, PLGA, polycaprolactone, poe, poly-anhydride, the polyphosphazene, its molecular weight is 10,000～100,000, and the hydrophilic block proportion is 4～20% in this amphipathic condensation material;
Described step 2) stabilizing agent of aqueous phase comprises one or more in polyvinyl alcohol, polyglyceryl fatty acid ester, Tween-81, polyoxyethylene sorbitol acid anhydride laurate and the dodecyl sodium sulfate, and stabilizer concentration is 0.1wt%～10wt%;
The aperture of microporous membrane is 0.5～200 μ m in the described step 3), and the pressure by microporous membrane is 1～2000kPa.
6. the preparation method of the porous microsphere for pharmaceutical carrier according to claim 5 is characterized in that,
In the described step 1), described organic solvent comprises one or more in dichloromethane, chloroform, Carbon bisulfide and the dimethylbenzene.
7. the preparation method of the porous microsphere for pharmaceutical carrier according to claim 5, it is characterized in that, described organic solvent comprises one or both in dichloromethane and the chloroform, and described two block amphipathic nature polyalcohol materials comprise the amphipathic condensation material of polylactic acid, PLGA or polycaprolactone and Polyethylene Glycol copolymerization gained.
8. a method of utilizing the described porous microsphere carrying medicament for pharmaceutical carrier of claim 1 is characterized in that, the mass ratio of pressing 1:2～100 mixes medicine with porous microsphere, and described medicine is to be selected from a kind of in polypeptide, the protide.
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