CN101849850A - Bionic in-situ regeneration repair nano sticking patch and preparation method and application thereof - Google Patents
Bionic in-situ regeneration repair nano sticking patch and preparation method and application thereof Download PDFInfo
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- CN101849850A CN101849850A CN201010144611A CN201010144611A CN101849850A CN 101849850 A CN101849850 A CN 101849850A CN 201010144611 A CN201010144611 A CN 201010144611A CN 201010144611 A CN201010144611 A CN 201010144611A CN 101849850 A CN101849850 A CN 101849850A
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Abstract
The invention relates to a bionic in-situ regeneration repair nano sticking patch, which is formed by overlapping an layer A with a cationic group material and a layer B with an anionic group material sequentially, wherein the sticking patch comprises 20 to 80 percent by mass of the A layer with the cationic group material and the balance of the layer B with the anionic group material; layer A with the cationic group material comprises at least one of polysaccharide with a cationic group, protein with the cationic group or opypeptide (polypeptide) with the cationic group; layer B with the anionic group material comprises at least one of polysaccharide with an anionic group, protein with the anionic group or opypeptide (polypeptide) with the anionic group; and the sticking patch comprises not more than 10 percent by mass of added functional factors and/or functional polypeptide. The raw materials used by the invention are materials which can be degraded in vitro and have biocompatibility. The sticking patch is flexible, has good tissue adhesivity, is suitable for concave-convex surfaces of visceral organs, has strong stretching resistance and air pressure resistance, is suitable for application of sticking, sealing, leak stoppage, hemostasia, isolation, repair, adhesion prevention and artificial meninges of defect tissues and can also be used as a slowly-releasing carrier of a medicament and a nano-grade tissue engineering support material.
Description
Technical field
The invention belongs to the biological medicine technology field, be specifically related to a kind of nano sticking patch with anionic group material, functional factor and/or functional polypeptide, band cation group material composite composition.And the preparation method of this nano sticking patch is provided and the pasting of defective tissue, sealing, leak stopping, hemostasis, isolation in surgical operation, repairs, prevent the purposes aspect adhesion, infection and the artificial meninges, also can be used as the slow-released carrier and the nanoscale tissue engineering bracket material of medicine.
Background technology
Often need be in surgical operation to the sealing of damaged and damaged tissue, repair and prevent adhesion, existing product has the film and the gel products of carboxymethyl cellulose, chitosan, polylactic acid, collagen and Fibrin Glue, these products to a certain extent to the sealing of damaged tissue, repair and prevent that adhesion from having played positive effect.But because its product performance of function that had many defective effects, as collagen protein sponge or film product thickness from 0.5mm to 5mm, cause product not soft, attach poor performance with tissue, product is met tissue fluid and is formed sticky shape easily, product loses hot strength, make the doctor of Clinical Surgery operate inconvenience,, must add cross-linking agent such as glutaraldehyde etc. for obtaining certain mechanical property, cross-linking agent also causes calcification to form except that certain tissue toxicity is arranged easily; The untoward reaction such as human allergy that the animal-based protein series products exists big security risk (as virus, pathogenic microorganism etc.) and protein macromolecule to cause; Carboxymethyl cellulose, chitosan series products mechanical strength are not enough, the vivo degradation time or too short or oversize; And the poly-lactic acid products degraded produces acid product or the like; Therefore, a kind of innovative product that overcomes existing film product defects of development has very great clinical value.
In the present invention dexterously biomaterial bionical, promote the nano combined technology of preparing of tissue repair function and biomaterial to combine, the physicochemical property of product is improved, soft, organize adhesion good, adapt to the concavo-convex of organ surface, stretch-proof and anti-air pressure performance are strong, can be used as the pasting of defective tissue, sealing, leak stopping, hemostasis, isolation, repair, prevent the purposes of adhesion, infection and artificial meninges aspect.Also can be used as the slow-released carrier and the nanoscale tissue engineering bracket material of medicine.
Summary of the invention
The objective of the invention is weak point, a kind of bionic in-situ regeneration repair nano sticking patch is provided according to above-mentioned existing product
,Preparation method and use, the present invention at first provides a kind of bionical composite Nano sticking patch that tissue defect is had the in-situ regeneration repairing effect; Next provides a kind of preparation method of this bionical composite Nano sticking patch; Moreover provide this bionical composite Nano sticking patch as the pasting of defective tissue, sealing, leak stopping, hemostasis, isolation, repair, prevent the purposes of adhesion and artificial meninges aspect.Also can be used as the slow-released carrier and the nanoscale tissue engineering bracket material of medicine.
The object of the invention realizes being finished by following technical scheme:
A kind of bionic in-situ regeneration repair nano sticking patch, the constituted mode that it is characterized in that described sticking patch is that the B layer by the A layer of band cation group material and band anionic group material superposes successively and forms, wherein said stack successively refers to, by the constituted mode of [(A+B) n+A] or [(B+A) n+B], wherein stacking fold n can be positive integer.
The thickness of described A layer or B layer is nanoscale.
The mass percent that the A layer of described band cation group material accounts for described sticking patch is 20%-80%, and all the other are the B layer of band anionic group material.
Described B layer is band anionic group material, but this group material has a biocompatible materials for vivo degradation, and described band anionic group material comprises at least a in band anionic group polysaccharide, band anionic group albumen or the band anionic group polypeptide (poly-peptide).
Described band anionic group polysaccharide includes but are not limited to one or more in carboxymethyl cellulose, carboxymethyl chitosan, hyaluronic acid, alginic acid, chondroitin sulfate and heparin polysaccharide and the derivant thereof, and some macromolecular materials and derivant such as polylactic acid, polyglycolic acid.
Described A layer is band cation group material, but this group material has a biocompatible materials for vivo degradation, and described band cation group material comprises at least a in band cation group polysaccharide, band cation group albumen or the band cation group polypeptide (poly-peptide).
Described band cation group polysaccharide includes but are not limited to chitosan and derivant thereof.
Described band cation group polypeptide (poly-peptide) includes but are not limited to polylysine and derivant thereof.
Described group albumen and polypeptide (poly-peptide) include but are not limited to polyampholyte collagen, elastin laminin and hydrolyzate thereof.
Contain the functional factor and/or the functional polypeptide that are no more than 10% mass percent in the described sticking patch.
Described functional polypeptide include but are not limited to contain-arginine-glycine-aspartic acid-polypeptide, contain-valine-glycine-valine-alanine-proline-glycine-polypeptide or contain-isoleucine-lysine-valine-alanine-valine-polypeptide in one or more mixture.
Described functional factor includes but are not limited to fibronectin (FN), laminin (LN), VEGF (VEGF), Fibrinogen (fg), epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor (TGFs), bone morphogenetic protein (BMPs), insulin like growth factor (IGF), platelet derived growth factor (PDGF), thrombin and molecular weight are less than one or more the mixture in 10000 the oligochitosan.
A kind of preparation method of bionic in-situ regeneration repair nano sticking patch is characterized in that described method adopts following steps:
1) the A solution of cation group material and the B solution of band anionic group material are with in preparation respectively;
2) go up spraying A or B solution, drying and forming-film at a flat board (plate material must be nontoxic, and water insoluble or sour, alkaline solution remove thermal source through degerming and handle); Spraying thereon or immersion B or A solution wash with water for injection again, replace spraying so respectively or soak A liquid or B liquid, wash with water for injection, and repetitive operation n-1 time sprays or immersion A or B solution, at last more thereon with water for injection washing, drying and forming-film; The thickness of controlling every layer of A or B is a nanoscale, makes the described nano sticking patch as claim 1-2;
3) interpolation of functional factor and polypeptide can be added on 1) in described A or the B solution, quality is 0% ~ 10% than concentration; Also can to prepare quality separately be the spraying of 0% ~ 10% solution C than concentration or be immersed in 2) the nano sticking patch surface that makes;
4) as developer, 1) preparation A and/or B solution in can add an amount of riboflavin;
Described nano sticking patch is used for the pasting of defective tissue, sealing, leak stopping, hemostasis, isolation, repairs, prevents the purposes of adhesion and artificial meninges aspect, also can be used for the slow-released carrier as medicine.
Advantage of the present invention is, the present invention is compound by bioabsorbable polymer material, the analog cell epimatrix, for the berthing of cell, grow, breed and vivosphere is provided and obtains nutrition, carry out metabolism, and the vivo degradation speed of the optimum organization controlled material by material; Being added with of functional factor and functional structure polypeptide helps sticking, stretching of cell, between the adjustable cell-matrix and cell-intercellular interaction, thereby realizes the multifunctional usage of material at aspects such as the reparation of damaged tissue, regeneration; Make the physicochemical property of product be beneficial to the adhesion and the gene expression of cell by alternately the superpose preparation method gained nanometer materials of polyelectrolyte of material, particularly strengthened the mechanical property of material, avoided the toxic and side effects of cross-linking agent.
The specific embodiment
By the following examples feature of the present invention and other correlated characteristic are described in further detail, so that technical staff's of the same trade understanding:
Embodiment 1. prepares one 12 cm diameter culture dishs, a built-in same diameter polymer material film (handling through washing, sterilization and depyrogenation), drying; Respectively the A solution of preparation band cation group material (the 1mg/ml chitosan, 0.1mol/L acetic acid, 0.2mol/LNaCl, pH=4) and the B liquid of band anionic group material (the 1mg/ml carboxymethyl chitosan, 0.15mol/LNaCl, pH=6); Get part B liquid and add functional polypeptide (1 * 10
-4Mg/ml) be mixed with C liquid, the functional polypeptide amino acid sequence is: lysine-lysine-cysteine-serine-arginine-glycine-aspartic acid-serine-cysteine-lysine-lysine; Above-mentioned solution is filled to respectively in the instantaneous spraying equipment of high pressure, at first toward the instantaneous spraying C of culture dish inner high voltage liquid, drying and forming-film; Then the instantaneous spraying A of high pressure liquid washes with water for injection; Then the instantaneous spraying B of high pressure liquid with the water for injection flushing, so alternately sprays A liquid, B liquid, flushing, repetitive operation 800 times; Follow the instantaneous spraying A of high pressure liquid again, wash with water for injection; The last instantaneous spraying C of high pressure liquid, with the water for injection flushing, drying is taken off film, and veneer washes with water for injection, and dried gets finished product.
Embodiment 2. prepares one 12 cm diameter culture dishs, a built-in same diameter polymer material film (handling through washing, sterilization and depyrogenation), drying; (0.2mol/LNaCl is pH=4) with the B liquid of being with the anionic group material (alginic acid of 2mg/ml, 0.15mol/LNaCl, pH=6) abbreviation B liquid for 2mg/ml chitosan, 0.1mol/L acetic acid for the A solution of preparation band cation group material respectively; Getting part B liquid adds epidermal growth factor (EGF) and (0.01mg/ml) is mixed with C liquid; Above-mentioned solution is filled to respectively in the instantaneous spraying equipment of high pressure, at first toward the instantaneous spraying C of culture dish inner high voltage liquid, drying and forming-film; Then the instantaneous spraying A of high pressure liquid washes with water for injection; Then the instantaneous spraying B of high pressure liquid with the water for injection flushing, so alternately sprays A liquid, B liquid, flushing, repetitive operation 200 times; Follow the instantaneous spraying A of high pressure liquid again, wash with water for injection; The last instantaneous spraying C of high pressure liquid, with the water for injection flushing, drying is taken off film, and veneer washes with water for injection, and dried gets finished product.
Embodiment 3. prepares one 12 cm diameter culture dishs, a built-in same diameter polymer material film (handling) through washing and sterilization depyrogenation, and the water for injection flushing is dry; Respectively preparation band cation group material (1mg/ml collagen, 0.1mol/L acetic acid, 0.2mol/LNaCl, pH=3.5) and the B liquid of band anionic group material (the 1mg/ml hyaluronic acid, 0.15mol/LNaCl, pH=6); Above-mentioned solution is filled to respectively in the instantaneous spraying equipment of high pressure, at first toward the instantaneous spraying A of culture dish inner high voltage liquid, drying, then the instantaneous spraying B of high pressure liquid with the water for injection flushing, so alternately sprays A liquid, B liquid, flushing, repetitive operation 500 times; The instantaneous spraying A of high pressure liquid more at last, with the water for injection flushing, drying is taken off film, and veneer washes with water for injection, and dried gets finished product.
Embodiment 4. prepares one 12 cm diameter culture dishs, a built-in same diameter polymer material film (handling through washing, sterilization and depyrogenation), drying; Respectively the A solution of preparation band cation group material (the 5mg/ml chitosan, 0.1mol/L acetic acid, 0.2mol/LNaCl, pH=4) and the B liquid of band anionic group material (the 1mg/ml hyaluronic acid, 0.15mol/LNaCl, pH=6); Get part B liquid and add thrombin (concentration: 2000IU/ml) be mixed with C liquid; Above-mentioned solution is filled to respectively in the instantaneous spraying equipment of high pressure, at first toward the instantaneous spraying C of culture dish inner high voltage liquid, drying and forming-film; Then the instantaneous spraying A of high pressure liquid washes with water for injection; Then the instantaneous spraying B of high pressure liquid with the water for injection flushing, so alternately sprays A liquid, B liquid, flushing, repetitive operation 300 times; Follow the instantaneous spraying A of high pressure liquid again, wash with water for injection; The last instantaneous spraying C of high pressure liquid, with the water for injection flushing, drying is taken off film, and veneer washes with water for injection, and dried gets finished product.
Embodiment 5. prepares one 12 cm diameter culture dishs, a built-in same diameter polymer material film (handling through washing, sterilization and depyrogenation), drying; A solution (1mg/ml polylysine, 0.1mol/L acetic acid, the 0.2mol/LNaCl of cation group material is with in preparation respectively, pH=4) and the B liquid (collagen of 1mg/ml of band anionic group material, the VEGF of 10ug/ml, 0.15mol/LNaCl pH=8) is called for short B liquid; At first toward the interior dip-coating B liquid of culture dish, drying and forming-film is followed dip-coating A liquid, washes with water for injection, so alternately dip-coating B liquid, A liquid, flushing, repetitive operation 100 times, dip-coating B liquid more at last, flushing, drying is taken off film, and veneer washes with water for injection, and dried gets finished product.
By the prepared sample of embodiment (to call test sample in the following text), biology and mechanical property evaluation test have been carried out in its safety and effectiveness:
1.Cell toxicity test:
Reference/technical standard: GB/T16886.5-2003
Cell strain: L-929 cell (l cell)
Culture fluid: contain 10%(V/V) MEM of calf serum
Blank: with batch cell culture fluid
Negative control: high density polyethylene (HDPE)
Positive control: 5g/L phenol solution
Lixiviate medium: with criticizing the MEM that does not contain calf serum
Extraction time: 24 hours
Test sample lixiviate ratio: 1g/5ml
Test method: lixiviating solution test (mtt assay)
At 27 ℃, 5%CO
2Blank, negative control, positive control and test sample lixiviating solution contact the cell of adherent growth, cultivate 72h after, add MTT liquid, hatch 4h.After the absorption, add DMSO, under wavelength 630nm, each group is carried out absorbance measurement, and calculate the relative rate of increase of cell by spectrophotometer.
Result: the cytotoxicity of test sample: 0 grade
Conclusion: with reference to GB/T16886.5-2003, this test sample no cytotoxicity.
2.The Intradermal irritant test
Reference/technical standard: GB/T16886.10-2005
Experimental animal: healthy new zealand rabbit
Lixiviate medium: 0.9% sodium chloride injection
Test sample: material lixiviating solution
Negative control: with batch lixiviate medium
Route of exposure: intradermal injection
Judging quota: observe 24h, 48h, 72h erythema, the edema extent of reaction
The result: injection back 24h, 48h, 72h injection site and surrounding skin are organized does not all have erythema, the edema reaction, and test side dermoreaction is not more than the control sides dermoreaction.
Conclusion: with reference to GB/T16886.10-2005, this test sample does not have the Intradermal irritant reaction.
3.Acute general toxicity test
Reference/technical standard: GB/T16886.11-1997/ASTMF750
Experimental animal: healthy mice
Lixiviate medium: 0.9% sodium chloride injection
Test sample: material lixiviating solution
Negative control: with batch lixiviate medium
Route of exposure: tail vein injection
Judging quota: observe 4h, 24h, 48h, 72h animal general state, toxicity performance and dead animal number.
The result: the reaction of test sample treated animal is not more than the negative control treated animal in the 72h observation period.
Conclusion: with reference to GB/T16886.11-1997/ASTMF750, the acute general toxicity result of the test of this test sample meets the requirements.
4.Hemolytic test
Reference/technical standard: GB/T16886.4-2003/GB/T16175-1996
Experimental animal: healthy new zealand rabbit
Dilution anticoagulant Sanguis Leporis seu oryctolagi preparation: fresh anticoagulant Sanguis Leporis seu oryctolagi+0.9% sodium chloride injection
Negative control: 0.9% sodium chloride injection
Positive control: distilled water
Route of exposure: tail vein injection
Test method: test sample is immersed in 0.9% sodium chloride injection by a certain percentage, behind 37 ℃ of water bath heat preservation 30min, add 0.2ml and dilute fresh anticoagulant Sanguis Leporis seu oryctolagi, 37 ℃ of water bath heat preservation 60min.Get supernatant behind the centrifugal 5min of 2500rpm, measure its absorbance at the 545nm place, calculate hemolysis rate with ultraviolet spectrophotometer.
The result: the hemolysis rate of this test sample is 0.2%.
Conclusion: with reference to GB/T16886.4-2003, the hemolytic test result of this test sample meets the requirement of medical material.
5.Mechanical property test
Reference/technical standard: M.J.Smith, M.J.McClure, S.A.Sell, etal.Suture-reinforcedelectrospunpolydioxanone – elastinsmall-diametertubesforuseinvasculartissueengineer ing:Afeasibilitystudy.ActaBiomaterialia, 2008,4 (1): 58 ~ 66
Test method: at the tensile property of the omnipotent mechanical test aircraft measurements of M350-20KN sample: pick off 250kgf, draw speed 10mm/min, sample size is 3.
The result: the hot strength of this test sample is 589 ~ 612KPa, and elongation at break maintains in the 140-160% scope.
Conclusion: reference literature, this test sample mechanical property satisfy the demand of human body interior mechanics environment fully.
Can be used for the damaged aspect of tissue by above evaluation test and data declaration product of the present invention, and safe and effective.
Below can further prove result of use of the present invention by animal experiment:
30 of new zealand rabbits, body weight (2500 ± 40) gram, male and female are regardless of, and are divided into two groups at random: A organizes 15 (being used for anatomic observation wound angiogenesis), and B organizes 15 (being used for anatomic observation wound repair situation); Be ready to specification and be 1.5X1.5 centimetre product of the present invention; All animals adopts the anesthesia of injection urethane, anaesthetizes safe back dorsal position, is fixed on the operating-table; Make the wound of 3 millimeters of diameters in the roughly the same position of each new zealand rabbit pulmonary; Product of the present invention is attached on the wound of 3 millimeters of experiment new zealand rabbit pulmonary diameters, sews up and dissect otch, postoperative sub-cage rearing, another day anatomic observation.
Result of the test: the intensity of product of the present invention can withstand the air pressure when breathing.Average 8 days, pulmonary's wound of A group experiment new zealand rabbit began to generate blood vessel, wound healing after B organizes average 27.5 days.Behind the wound healing, product of the present invention is natural degradation in vivo.Product of the present invention can adapt to the concavo-convex of organ surface, can touch wound, and not can with other tissue adhesions.Product of the present invention can be used for softness and internal organs that are difficult to sew up or easily hemorrhage internal organs such as stomach, liver, helps to shorten the surgical operation time, and shortens rehabilitation duration.
Claims (14)
1. bionic in-situ regeneration repair nano sticking patch, the constituted mode that it is characterized in that described sticking patch is that the B layer by the A layer of band cation group material and band anionic group material superposes successively and forms, wherein said stack successively refers to, by the constituted mode of [(A+B) n+A] or [(B+A) n+B], wherein stacking fold n can be positive integer.
2. a kind of bionic in-situ regeneration repair nano sticking patch according to claim 1, the thickness that it is characterized in that described A layer or B layer is nanoscale.
3. a kind of bionic in-situ regeneration repair nano sticking patch according to claim 1, the mass percent that the A layer that it is characterized in that described band cation group material accounts for described sticking patch is 20%-80%, all the other are the B layer of band anionic group material.
4. a kind of bionic in-situ regeneration repair nano sticking patch according to claim 1 and 2, it is characterized in that described B layer is band anionic group material, but this group material has a biocompatible materials for vivo degradation, and described band anionic group material comprises at least a in band anionic group polysaccharide, band anionic group albumen or the band anionic group polypeptide (poly-peptide).
5. a kind of bionic in-situ regeneration repair nano sticking patch according to claim 4, it is characterized in that described band anionic group polysaccharide includes but are not limited to one or more in carboxymethyl cellulose, carboxymethyl chitosan, hyaluronic acid, alginic acid, chondroitin sulfate and heparin polysaccharide and the derivant thereof, and some macromolecular materials and derivant such as polylactic acid, polyglycolic acid.
6. a kind of bionic in-situ regeneration repair nano sticking patch according to claim 1 and 2, it is characterized in that described A layer is band cation group material, but this group material has a biocompatible materials for vivo degradation, and described band cation group material comprises at least a in band cation group polysaccharide, band cation group albumen or the band cation group polypeptide (poly-peptide).
7. a kind of bionic in-situ regeneration repair nano sticking patch according to claim 6 is characterized in that described band cation group polysaccharide includes but are not limited to chitosan and derivant thereof.
8. a kind of bionic in-situ regeneration repair nano sticking patch according to claim 6 is characterized in that described band cation group polypeptide (poly-peptide) includes but are not limited to polylysine and derivant thereof.
9. according to claim 4 or 6 described a kind of bionic in-situ regeneration repair nano sticking patch, it is characterized in that described group albumen and polypeptide (poly-peptide) include but are not limited to polyampholyte collagen, elastin laminin and hydrolyzate thereof.
10. a kind of bionic in-situ regeneration repair nano sticking patch according to claim 3 is characterized in that containing in the described sticking patch functional factor and/or the functional polypeptide that is no more than 10% mass percent.
11. a kind of bionic in-situ regeneration repair nano sticking patch according to claim 10, it is characterized in that, described functional polypeptide be include but are not limited to contain-arginine-glycine-aspartic acid-polypeptide, contain-valine-glycine-valine-alanine-proline-glycine-polypeptide or contain-isoleucine-lysine-valine-alanine-valine-polypeptide in one or more mixture.
12. a kind of bionic in-situ regeneration repair nano sticking patch according to claim 10, it is characterized in that described functional factor includes but are not limited to fibronectin (FN), laminin (LN), Fibrinogen (fg), VEGF (VEGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor (TGFs), bone morphogenetic protein (BMPs), insulin like growth factor (IGF), platelet derived growth factor (PDGF), thrombin, molecular weight is less than one or more the mixture in 10000 the oligochitosan.
13. the preparation method of a bionic in-situ regeneration repair nano sticking patch is characterized in that described method adopts following steps:
(1) the A solution of cation group material and the B solution of band anionic group material are with in preparation respectively;
(2) spraying A or B solution on a flat board, drying and forming-film, wherein plate material must be nontoxic, and water insoluble or sour, alkaline solution remove thermal source through degerming and handle; Spraying thereon or immersion B or A solution wash with water for injection again, replace spraying so respectively or soak A liquid or B liquid, wash with water for injection, and repetitive operation n-1 time sprays or immersion A or B solution, at last more thereon with water for injection washing, drying and forming-film; The thickness of controlling every layer of A or B is a nanoscale, makes the described nano sticking patch as claim 1-2;
(3) can add functional factor and polypeptide in (1) described A or B solution, mass percent concentration is 0%-10%; Also can prepare functional factor and polypeptide mass percent concentration separately and be the solution C spraying of 0%-10% or be immersed in the nano sticking patch surface that (2) make.
14. according to the described a kind of bionic in-situ regeneration repair nano sticking patch of claim 1-2, it is characterized in that described nano sticking patch is used for the pasting of defective tissue, sealing, leak stopping, hemostasis, isolation, repairs, prevents the purposes of adhesion and artificial meninges aspect, also can be used for slow-released carrier and nanoscale tissue engineering bracket material as medicine.
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