CN100349623C - Active gliadin porous stent preparation method - Google Patents
Active gliadin porous stent preparation method Download PDFInfo
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- CN100349623C CN100349623C CNB2006100260860A CN200610026086A CN100349623C CN 100349623 C CN100349623 C CN 100349623C CN B2006100260860 A CNB2006100260860 A CN B2006100260860A CN 200610026086 A CN200610026086 A CN 200610026086A CN 100349623 C CN100349623 C CN 100349623C
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Abstract
The present invention relates to an active gliadin porous stent preparation method in the technical field of bioengineering. The preparation method comprises the followinng steps: gliadin microballoons with active factors or direct gliadin and the active factors are uniformly mixed according to the weight ratio of 1: 10 to 560 at-40 DEG C to 0 DEG C, and then the mixture is mixed with ice particles according to the ratio of 1: 0.1 to 5; compression moulding is carried out at-40 to 0 DEG C, and the ice particles in a stent are removed by freeze drying; the gliadin porous stent coated with the active factors is formed for 1 h to 10 days under the conditions of 10 to 75 DEG C and humidity of 60 to 100 %; water in the stent is removed by freeze drying. The present invention can obtain the active gliadin porous stent with proper porosity and controllable content of the pore size and the active factors. The active factors maintain good bioactivity and can be slowly released. When the active gliadin porous stent is used as a stent material and is applied to the tissue engineering field, the active gliadin porous stent can act for a long time.
Description
Technical field
The present invention relates to a kind of method of technical field of bioengineering, specifically, what relate to is a kind of preparation method of active gliadin porous stent.
Background technology
Tissue engineering bracket material is one of emphasis of biomedical sector Tissue Engineering Study; Biomaterial is the three-dimensional rack that the seed cell formative tissue is depended on for existence and depended on before, it can be with cell fixation in certain position, for physiological activities such as its growth, breeding, metabolism and extracellular matrix secretion provide the place, and guiding regenerating tissues basic configuration.Timbering material as organizational project should possess following characteristics: 1. good biocompatibility: be beneficial to cell adhesion and propagation, and no cytotoxicity, non-immunogenicity, reaction does not cause inflammation; 2. biodegradability: can degrade fully, degradation rate forms the speed basically identical with tissue; 3. have plasticity and certain mechanical strength, bootable tissue regeneration can be kept specific size and shape in the tissue regeneration process; 4. certain porosity, suitably big or small aperture and connectivity, cell can be evenly distributed on material surface and inside, can obtain enough nutrition, and growing into for blood vessel provides support; 5. material and catabolite thereof nontoxic, body is had no adverse reaction, stable in properties is easily stored, easily sterilization etc.
The mechanical property of biomaterial is an organizational project, particularly one of key factor in the hard tissue engineering.At present, natural biologic material has been subjected to extensive studies, comprises chitin, fibrin, collagen protein, chitosan etc.These material common features are exactly degradable, good biocompatibility, but the three-dimensional rack mechanical strength deficiency of preparation, thus limited their development.Zein is the main storage protein of corn, accounts for the 45%-50% of corn total protein.Zein can be processed into resin, has good thermoplastic, tenacity, hydrophobicity, degradability and antibiotic property etc.Zein has been studied and has been used for adhesive of medical, biodegradable plastic, chewing gum, the packaging for foodstuff of edible moisture-resisting, acid-sensitive sense drug delivery system, microencapsulation insecticide, oral drugs odor mask, many aspects such as biological experiment band and wound dressing.Studies show that: zein has good biodegradability and biocompatibility, is a kind of new type natural polymer that can be applicable to organizational project.
Find through literature search prior art, the Chinese patent publication number is patents such as CN1555892A, all be that Phytogenous alcohol-soluble protein is mixed with porogen such as Sal, natural molding under the condition of uniform temperature and humidity, or remove the porogen postlyophilization and make through compression molding, decocting in water.Although said method can obtain to have the support of certain mechanical strength and hole characteristic, and can make the rack bearing active factors by methods such as immersions; But, can't effectively control the higher activity of content, deenergized period and maintenance of active factors in the support as required because be subject to processing the restriction of condition.
Summary of the invention
The objective of the invention is to overcome above-mentioned deficiency of the prior art and defective, a kind of preparation method of active gliadin porous stent is provided, bioactie agent content is controlled in the feasible support of preparing, and is evenly distributed; Because support is to be prepared under the low temperature temperate condition, active component keeps than high bioactivity, and can slowly discharge.When being applied to field of tissue engineering technology as timbering material, long-term role in vivo.
The present invention is achieved by the following technical solutions; the present invention mixes alcohol soluble protein and active factors with the ice granule; perhaps with the alcohol soluble protein microsphere of the carrying active factor and ice granule mixing aftershaping; its pore size can realize that porosity can be regulated by control albumen and ice proportion of particles by regulation and control ice granular size.
Concrete steps of the present invention are as follows:
1. at-40 ℃-0 ℃, with the alcohol soluble protein microsphere of the bag carrying active factor, perhaps directly active factors and alcohol soluble protein are pressed 1: after the weight ratio of 10-560 is mixed, the sealing pre-cooling, with ice granule by 1: the weight ratio of 0.1-5 is mixed, and utilizes the compression moulding molding at-40 ℃-0 ℃.
2. will be 1. the gliadin porous stent (promptly by icing the support that granule, active factors and alcohol soluble protein mixing are formed) of the alcohol soluble protein microsphere porous scaffolds of the ice granule of molding-Bao carrying active factor (promptly the alcohol soluble protein microsphere by the ice granule and the bag carrying active factor mixes the support of being formed) or ice granule-Bao carrying active factor utilize freeze-drying to remove ice granule in the support.
3. will be 2. the alcohol soluble protein microsphere porous scaffolds of the bag carrying active factor after the lyophilization or the gliadin porous stent of the bag carrying active factor be placed on 10 ℃-75 ℃, the molding in-10 days in following 1 hour of 60%-100% humidity, utilize lyophilization to remove moisture in the support again, obtain active gliadin porous stent.
Described alcohol soluble protein is the Phytogenous alcohol-soluble protein of Semen Tritici aestivi, Fructus Hordei Vulgaris, naked barley, Herba bromi japonici or corn etc.
Described alcohol is C
1-4Aliphatic alcohol; Described aliphatic alcohol is further defined to: ethanol.
The alcohol soluble protein microsphere of the described bag carrying active factor, its preparation method is: under 20 ℃-37 ℃, with alcohol soluble protein with contain the aqueous solution of 60%-95% alcohol and active factors molten altogether after, the adjusting determining alcohol is 10%-50%, is prepared into the alcohol soluble protein microsphere that includes active factors.The entrapment efficiency of the alcohol soluble protein microsphere of the bag carrying active factor that is obtained by this method is 0.2%-40%.
Described active gliadin porous stent, porosity is: 30%-85%, pore size is: 50 μ m-1000 μ m; Mainly be to be used for field of tissue engineering technology.
Described active factors is that activity is subjected to temperature and the destructive somatomedin of organic solvent easily.
The present invention adopts the ice granule as perforating agent, and the gliadin porous stent of the bag carrying active factor that obtains is to finish below 0 ℃ basically, and 10 ℃-75 ℃ institute's time of staying are of short duration; In addition, whole process of preparation is except using ethanol, do not relate to other organic solvents, and do not need processing such as decocting in water, so both got rid of the organic solvent residue toxicity problem, got rid of temperature and organic solvent destruction again, thereby the activity of active factors is at utmost kept active factors.Because the present invention prepares the incipient stage, adopted active factors and albumen directly have been mixed in proportion, perhaps directly use the alcohol soluble protein microsphere of the bag carrying active factor that active factors content determines, therefore, better controlled the content of active factors in the active gliadin porous stent.When material uses, control the concentration of local active factors whereby in field of tissue engineering technology, prevent the excessive concentration toxigenicity, cross the low deficiency that can't play effectiveness.Secondly, active factors and alcohol soluble protein are evenly distributed in the support, have avoided the conventional support of preparation earlier, and when adopting method medicine carryings such as soaking absorption again, active factors can't go deep into internal stent, and cause relatively short disadvantage deenergized period thus; Simultaneously, the gliadin porous stent of the bag carrying active factor can be along with proteic degraded, and the active factors of internal stent is discharged gradually, the performance long-acting.
Description of drawings
Fig. 1: the bag of phase separation method preparation is loaded with the alcohol soluble protein microsphere of dexamethasone sodium phosphate, a: raw material (scale=74 μ m), b: the alcohol soluble protein microsphere (scale=1.18 μ m) of the bag carrying active factor.
Fig. 2: the ice granule is as the active gliadin porous stent of perforating agent preparation, and A: bag is loaded with the alcohol soluble protein microsphere porous scaffolds (scale=141 μ m) of dexamethasone sodium phosphate; B: bag is loaded with the gliadin porous stent (scale=139 μ m) of dexamethasone sodium phosphate.
Fig. 3: the ice granule is loaded with the release in vitro kinetics of dexamethasone sodium phosphate in the alcohol soluble protein microsphere porous scaffolds of dexamethasone sodium phosphate and the gliadin porous stent that bag is loaded with dexamethasone sodium phosphate as the bag of perforating agent preparation.
The specific embodiment
In conjunction with the accompanying drawings and technical solution of the present invention following examples are provided:
Embodiment 1:
Porosity is the preparation that 44% bag is loaded with the alcohol soluble protein microsphere porous scaffolds and the gliadin porous stent that bag is loaded with dexamethasone sodium phosphate of dexamethasone sodium phosphate:
1. bag is loaded with the alcohol soluble protein microsphere of dexamethasone sodium phosphate, after perhaps directly dexamethasone sodium phosphate and alcohol soluble protein being mixed by 1: 10 weight ratio,-40 ℃ of sealing pre-coolings, after the ice granule mixes by 1: 4.22 weight ratio, utilize the rustless steel grinding tool pressing mold of pre-cooling to make 5 * 5 * 5mm at-40 ℃
3Cylindrical body.
2. the ice granule-Bao of molding is loaded with the alcohol soluble protein microsphere porous scaffolds of dexamethasone sodium phosphate or the gliadin porous stent that ice granule-Bao is loaded with dexamethasone sodium phosphate, utilizes freeze-drying to remove ice granule in the support.
3. the bag after the lyophilization is loaded with the alcohol soluble protein microsphere porous scaffolds of dexamethasone sodium phosphate or gliadin porous stent that bag is loaded with dexamethasone sodium phosphate and is placed on 75 ℃, following 1 hour aftershaping of 100% humidity, moisture is removed in lyophilization, as Fig. 2.
Wrapping the alcohol soluble protein microsphere preparation that is loaded with dexamethasone sodium phosphate in the foregoing description realizes by the following method:
Alcohol soluble protein and dexamethasone sodium phosphate be dissolved in respectively in 60% the ethanol water, behind the mix homogeneously, adjust concentration of alcohol to 40% rapidly, obtain wrapping the alcohol soluble protein microsphere suspension that is loaded with dexamethasone sodium phosphate, lyophilization must be wrapped the rough powder of alcohol soluble protein microsphere that is loaded with dexamethasone sodium phosphate.After metal spraying is handled, sem observation microsphere form and particle size distribution, result as shown in Figure 1, prepared protein microsphere diameter is controlled between the 40nm-100nm.According to this method,, regulate and control active factors content in the support, and further control its fine distribution and release in support by active factors content in the control microsphere.Well avoided in the conventional active support preparation process, active component only sticks to the surface in support or hole, has prolonged active factors release time greatly and has regulated burst size.
According to the active gliadin porous stent that the foregoing description finally obtains, its content determination of Dexamethasone Sodium is: 9.1%, and porosity is: 44.4% ± 9.0%, pore size is 150 μ m-250 μ m, the connectivity in hole is good, as Fig. 2.Whole process was stagnated 1 hour under 100% humidity only at 75 ℃, and active factors is active to keep good.
Porosity is the preparation that 65% bag is loaded with the alcohol soluble protein microsphere porous scaffolds and the gliadin porous stent that bag is loaded with dexamethasone sodium phosphate of dexamethasone sodium phosphate:
1. bag is loaded with the alcohol soluble protein microsphere of dexamethasone sodium phosphate, after perhaps directly dexamethasone sodium phosphate and alcohol soluble protein being mixed by 1: 560 weight ratio,-20 ℃ of sealing pre-coolings, after the ice granule mixes by 1: 1.26 weight ratio, utilize the rustless steel grinding tool pressing mold of pre-cooling to make 5 * 5 * 5mm at-20 ℃
3Cylindrical body.
2. the ice granule-Bao of molding being loaded with the alcohol soluble protein microsphere porous scaffolds of dexamethasone sodium phosphate or gliadin porous stent that ice granule-Bao is loaded with dexamethasone sodium phosphate utilizes freeze-drying to remove ice granule in the support.
3. the bag after the lyophilization is loaded with the alcohol soluble protein microsphere porous scaffolds of dexamethasone sodium phosphate or gliadin porous stent that bag is loaded with dexamethasone sodium phosphate and is placed on 10 ℃, following 10 days aftershapings of 60% humidity, moisture is removed in lyophilization, as Fig. 2.
Wrapping the alcohol soluble protein microsphere preparation that is loaded with dexamethasone sodium phosphate in the foregoing description realizes by the following method:
Alcohol soluble protein and dexamethasone sodium phosphate be dissolved in respectively in 60% the ethanol water, behind the mix homogeneously, adjust concentration of alcohol to 40% rapidly, obtain wrapping the alcohol soluble protein microsphere suspension that is loaded with dexamethasone sodium phosphate, lyophilization must be wrapped the rough powder of alcohol soluble protein microsphere that is loaded with dexamethasone sodium phosphate.After metal spraying is handled, sem observation microsphere form and particle size distribution, result as shown in Figure 1, prepared protein microsphere diameter is controlled between the 40nm-100nm.According to this method,, regulate and control active factors content in the support, and further control its fine distribution and release in support by active factors content in the control microsphere.Well avoided in the conventional active support preparation process, active component only sticks to the surface in support or hole, has prolonged active factors release time greatly and has regulated rate of release.
According to the active gliadin porous stent that the foregoing description finally obtains, its content determination of Dexamethasone Sodium is: 0.2%, and porosity is: 65.0% ± 4.0%, pore size is 150 μ m-250 μ m, the connectivity in hole is good, as Fig. 2.Whole process was stagnated 10 days under 60% humidity only at 10 ℃, and active factors is active to keep good.
Embodiment 3:
Porosity is the preparation that 80% bag is loaded with the alcohol soluble protein microsphere porous scaffolds and the gliadin porous stent that bag is loaded with dexamethasone sodium phosphate of dexamethasone sodium phosphate:
1. bag is loaded with the alcohol soluble protein microsphere of dexamethasone sodium phosphate, after perhaps directly dexamethasone sodium phosphate and alcohol soluble protein being mixed by 1: 56 weight ratio, 0 ℃ of sealing pre-cooling, after the ice granule mixes by 1: 0.24 weight ratio, utilize the rustless steel grinding tool pressing mold of pre-cooling to make 5 * 5 * 5mm at 0 ℃
3Cylindrical body.
2. the ice granule-Bao of molding being loaded with the alcohol soluble protein microsphere porous scaffolds of dexamethasone sodium phosphate or gliadin porous stent that ice granule-Bao is loaded with dexamethasone sodium phosphate utilizes freeze-drying to remove ice granule in the support.
3. the bag after the lyophilization is loaded with the alcohol soluble protein microsphere porous scaffolds of dexamethasone sodium phosphate or gliadin porous stent that bag is loaded with dexamethasone sodium phosphate and is placed on 37 ℃, following 5 days aftershapings of 80% humidity, moisture is removed in lyophilization, as Fig. 2.
Wrapping the alcohol soluble protein microsphere preparation that is loaded with dexamethasone sodium phosphate in the foregoing description obtains by the following method:
Alcohol soluble protein and dexamethasone sodium phosphate be dissolved in respectively in 60% the ethanol water, behind the mix homogeneously, adjust concentration of alcohol to 40% rapidly, obtain wrapping the alcohol soluble protein microsphere suspension that is loaded with dexamethasone sodium phosphate, lyophilization must be wrapped the rough powder of alcohol soluble protein microsphere that is loaded with dexamethasone sodium phosphate.After metal spraying is handled, sem observation microsphere form and particle size distribution, result as shown in Figure 1, prepared protein microsphere diameter is controlled between the 40nm-100nm.According to this method,, regulate and control active factors content in the support, and further control its fine distribution and release in support by active factors content in the control microsphere.Well avoided in the conventional active support preparation process, active component only sticks to the surface in support or hole, has prolonged active factors release time greatly and has regulated rate of release.
According to the active gliadin porous stent that the foregoing description finally obtains, its content determination of Dexamethasone Sodium is: 1.8%, and porosity is: 80.3% ± 3.7%, pore size is 150 μ m-250 μ m, the connectivity in hole is good, as Fig. 2.Whole process was stagnated 5 days under 80% humidity only at 37 ℃, and active factors is active to keep good.
Table: the ice granule as the active gliadin porous stent porosity of perforating agent preparation with ice granule-Protein ratios variation diagram (n=3)
| Grouping | 1 | 2 | 3 | 4 | 5 |
| Alcohol soluble protein: ice granule (w/w) | 4.22 | 1.26 | 0.52 | 0.34 | 0.24 |
| Theoretical porosity (%) | 24.01 | 50.64 | 71.24 | 79.17 | 84.5 |
| True porosity (%) | 44.37± 8.99 | 64.98± 4.03 | 76.84± 1.18 | 81.79± 0.93 | 80.25± 3.67 |
Embodiment 4:
Bag is loaded with the release in vitro kinetics of active factors in the alcohol soluble protein microsphere porous scaffolds of dexamethasone sodium phosphate and the gliadin porous stent that bag is loaded with dexamethasone sodium phosphate
The bag of preparation is loaded with the alcohol soluble protein microsphere porous scaffolds of dexamethasone sodium phosphate and the gliadin porous stent that bag is loaded with dexamethasone sodium phosphate, be immersed in 1.2mL phosphate buffer (PBS respectively, pH7.2,37 ℃) in, regularly replace fresh PBS solution, and utilize spectrophotography in 240nm detection of active factor content, calculate rate of release.The result as shown in Figure 3, bag is loaded with in the alcohol soluble protein microsphere porous scaffolds of dexamethasone sodium phosphate and the gliadin porous stent that bag is loaded with dexamethasone sodium phosphate active factors in in-vitro simulated internal milieu, active factors was kept 35 days release time, drug release two has reached 85% and 95% respectively, still has the part active factors not discharge.In vivo, along with the effect of hydrolytic enzyme in the body, albumen is degraded gradually, and active factors will discharge gradually, thereby keeps the more macrocyclic release of medicine.
Claims (3)
1, a kind of preparation method of active gliadin porous stent is characterized in that, concrete steps are as follows:
1. at-40 ℃-0 ℃, with the alcohol soluble protein microsphere of the bag carrying active factor, perhaps directly alcohol soluble protein and active factors are pressed 1: behind the weight ratio mix homogeneously of 10-560, the sealing pre-cooling, with ice granule by 1: 0.1-5 mixes, and utilizes the compression moulding molding at-40 ℃-0 ℃;
The alcohol soluble protein microsphere of the described bag carrying active factor, its preparation method is: under 20 ℃-37 ℃, with alcohol soluble protein with contain the aqueous solution of 60%-95% alcohol and active factors molten altogether after, the adjusting determining alcohol is 10%-50%, is prepared into the alcohol soluble protein microsphere that includes active factors; Described alcohol is C
1-4Aliphatic alcohol;
2. the alcohol soluble protein microsphere porous scaffolds of the gliadin porous stent of the ice granule-Bao carrying active factor of molding and ice granule-Bao carrying active factor is utilized freeze-drying to remove ice granule in the support;
The alcohol soluble protein support that 3. will wrap the carrying active factor is placed on 10 ℃-75 ℃, the molding in-10 days in following 1 hour of 60%-100% humidity, moisture is removed in lyophilization, the active gliadin porous stent porosity that obtains is: 30%-85%, pore size is: 50 μ m-1000 μ m.
2, the preparation method of active gliadin porous stent according to claim 1 is characterized in that: described alcohol soluble protein is the Phytogenous alcohol-soluble protein of Semen Tritici aestivi, Fructus Hordei Vulgaris, naked barley, Herba bromi japonici or corn.
3, the preparation method of active gliadin porous stent according to claim 1 is characterized in that: described aliphatic alcohol is ethanol.
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| CNB2006100260860A CN100349623C (en) | 2006-04-27 | 2006-04-27 | Active gliadin porous stent preparation method |
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| CNB2006100260860A CN100349623C (en) | 2006-04-27 | 2006-04-27 | Active gliadin porous stent preparation method |
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| CN113679893A (en) * | 2021-10-14 | 2021-11-23 | 河南科技学院 | Preparation method of double-effect anticoagulant antibacterial artificial catheter |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6103255A (en) * | 1999-04-16 | 2000-08-15 | Rutgers, The State University | Porous polymer scaffolds for tissue engineering |
| CN1555892A (en) * | 2004-01-08 | 2004-12-22 | 上海交通大学 | Preparation method of plant origion alcohrl soluble protein three dimentional support |
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2006
- 2006-04-27 CN CNB2006100260860A patent/CN100349623C/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6103255A (en) * | 1999-04-16 | 2000-08-15 | Rutgers, The State University | Porous polymer scaffolds for tissue engineering |
| CN1555892A (en) * | 2004-01-08 | 2004-12-22 | 上海交通大学 | Preparation method of plant origion alcohrl soluble protein three dimentional support |
Non-Patent Citations (3)
| Title |
|---|
| 玉米醇溶蛋白微球制备条件的探索以及体外释药行为研究 孙庆恩等.黑龙江畜牧兽医，第10期 2005 * |
| 聚乳酸多孔支架制备及细胞实验 郭超等.中国生物医学工程学报，第24卷第1期 2005 * |
| 骨组织工程用PLGA多孔支架的制备及细胞毒性研究 孙浩等.化工时刊，第19卷第10期 2005 * |
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