A kind of polyvinyl alcohol hydrogel and its preparation method and applicationTechnical field
The present invention relates to a kind of polyvinyl alcohol hydrogels and its preparation method and application, belong to macromolecule hydrogel technology neckDomain.
Background technique
Skin is located at human body surface, is one of human body maximum, most important organ.Since the long-term large area of skin is exposed toIn external environment, the moment may all be damaged.When wound is extremely serious, such as burn, severe frostbite, mechanical trauma, slowlyUlcer caused by property disease etc., not only brings pain to patient, or even also result in death.Compared to traditional dressing, research and developmentPreparing, there is the new pattern compress for promoting Healing to be very necessary.
Polyvinyl alcohol (PVA) is a kind of long-chain water soluble polymer acted on by hydrolysing polyvinyl esters, nontoxic nothingTaste will not cause skin allergy to no skin irritation, and have good film forming and cohesive force, under certain condition may be usedTo form the good hydrogel of biocompatibility.
Winter Ling Su is a kind of active diterpenoids compound that separating-purifying comes out from Rabdosia rubescens, research shows that winter Ling Su hasStandby antibacterial, anti-inflammatory, analgesia, antitumor and other effects, by the extensive concern of field of biomedicine.However, winter Ling Su itself compared withThe water solubility of difference seriously affects its drug effect.In order to expand the application of winter Ling Su clinically, its utilization ratio of drug, research and development system are improvedA kind of standby new drug carrier is particularly important.
Sodium alginate is the natural biological macromolecular sodium salt extracted from natural brown alga, has cytotoxic and goodBiocompatibility.Resulting microballoon is prepared by sodium alginate and is widely used in biomedicine field.
Polyethyleneimine (PEI) has become non-viral genoid carrier field most researching value and application prospectA kind of gene transmission vector.However, simple polyethyleneimine still remains many defects, if cytotoxicity is relatively large, gatherClose object non-degradable etc..Therefore.Using polyethyleneimine as precursor structure, further modification overcomes disadvantages described above, synthesisPolyethylenimine derivates are used for the carrier of Microrna, are a kind of novel gene vector materials with development potentiality.
Microrna (microRNA, miR) is that one kind of discovered in recent years is about the non-coding single stranded RNA of 22nt, can silencingTo play regulatory function, miR almost participates in internal all bioelectric detecting processes, cooperates gene for the gene expression of post-transcriptional levelGene therapy may be implemented in carrier.
Summary of the invention
There is provided it is an object of the invention to overcome in place of above-mentioned the deficiencies in the prior art a kind of polyvinyl alcohol hydrogel andPreparation method and application, it is derivative which loads winter Ling Su/sodium alginate micro ball compound, Microrna/polyethyleneimineObject carrier has drug slow release function, can be improved the drug availability of winter Ling Su, and have the effect of gene therapy.
To achieve the above object, the technical scheme adopted by the invention is as follows: a kind of preparation method of polyvinyl alcohol hydrogel, packetInclude following steps:
(1) it configures polyvinyl alcohol water solution: stirring and dissolving in deionized water is added in polyvinyl alcohol, obtains polyvinyl alcohol waterSolution；
(2) winter Ling Su preparation load winter Ling Su/sodium alginate micro ball compound: is added to sodium alginate micro ball solutionIn, freeze-dried back；
(3) it prepares polyethylenimine derivates carrier: polyethyleneimine is added drop-wise in hyaluronic acid solution, obtain poly- secondAlkene imine derivative carrier；
(4) miR-29A solution preparation load miR-29A/ polyethylenimine derivates carrier: is added drop-wise to step (3) instituteIn the polyethylenimine derivates carrier obtained, freezen protective；
(5) it prepares polyvinyl alcohol hydrogel: step (1), step (2), step (4) obtained component being mixed, poly- second is obtainedEnol hydrogel.
Preparation method of the invention is by winter Ling Su/sodium alginate micro ball compound and Microrna/polyethylenimine derivatesCarrier is loaded into polyvinyl alcohol hydrogel material jointly, achievees the effect that medicament slow release and gene therapy, is conducive to push newThe research application of type dressing.
The preferred embodiment of preparation method as polyvinyl alcohol hydrogel of the present invention in the step (1), is gatheredThe mass concentration of vinyl alcohol aqueous solution is 5~20%, and solution temperature is 80~90 DEG C, and mixing speed is 300~500r/min.
The preferred embodiment of preparation method as polyvinyl alcohol hydrogel of the present invention, in the step (2), seaMosanom microballoon the preparation method comprises the following steps: emulsifier and vegetable oil are mixed to obtain mixed liquor, stirring at normal temperature, by sodium alginate aqueous solutionIt is added drop-wise in mixed liquor, continues stirring at normal temperature, calcium chloride solution is added and is stirred overnight, is then distinguished with isopropanol and deionized waterThree times, freeze-drying obtains sodium alginate micro ball to centrifuge washing.
The preferred embodiment of preparation method as polyvinyl alcohol hydrogel of the present invention, in the step (2), seaThe concentration of mosanom microspheres solution is 5~10mg/mL, and 0.5~2mg of winter Ling Su is added in every milliliter of sodium alginate micro ball solution, coldFreezing drying temperature is -60 DEG C, and sublimation drying is for 24 hours.
The preferred embodiment of preparation method as polyvinyl alcohol hydrogel of the present invention in the step (3), is gatheredEthylenimine derivatives carrier the preparation method comprises the following steps: using deionized water as solvent, configuration quality score be 0.5~2% it is transparent0.1~0.5g 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide (EDC), 0.2 is added in matter acid solution, every gram of hyaluronic acidEDC/NHS priming reaction occurs for~1g n-hydroxysuccinimide (NHS), 1~5g polyethyleneimine, and stirring at normal temperature is overnight,Dialysis three days is carried out with the bag filter that molecular cut off is 10K, is changed daily water 2~3 times, is finally freeze-dried spare, freezing is dryDry temperature is -60 DEG C, and sublimation drying is for 24 hours, to obtain polyethylenimine derivates carrier.
The preferred embodiment of preparation method as polyvinyl alcohol hydrogel of the present invention, in the step (4),The dosage of miR-29A is that 50~100ng miR-29A is added in every milligram of polyethylenimine derivates carrier.
MiR-29A of the present invention is to be made of endogenic non-coding nucleotide, have to the expression of protein molecularThere is the single-stranded microRNA s of adjusting function, research shows that the miR has at the relevant transcription factor of blood vessel and signaling moleculeImportant adjustment effect, it is expected that being applied in skin repair.
The preferred embodiment of preparation method as polyvinyl alcohol hydrogel of the present invention in the step (5), is gatheredPolyvinyl alcohol hydrogel the preparation method comprises the following steps: step (1), step (2), step (4) obtained component are mixed in equal volume, pour into moldIn, it is placed in refrigerated overnight at -20 DEG C.
Second aspect, the present invention provides a kind of polyvinyl alcohol hydrogel, the polyvinyl alcohol hydrogel is according to above-mentionedMade from preparation method.
The third aspect, the present invention provides above-mentioned polyvinyl alcohol hydrogel answering in the dressing of preparation treatment skin woundWith.
Compared with prior art, the invention has the benefit that hydrogel load winter Ling Su/sodium alginate of the invention is micro-Ball compound, Microrna/polyethylenimine derivates carrier, have the function of medicament slow release and micro RNA regulation and control, Neng GoutiThe drug availability of high winter Ling Su, and have the effect of gene therapy, it can be applied in skin wound dressing.
Detailed description of the invention
Fig. 1 is the sodium alginate micro ball scanning electron microscope (SEM) photograph of embodiment 1.
Fig. 2 is the polyethylenimine derivates carrier core magnetic chart of embodiment 1.
Fig. 3 is the hydrogel material scanning electron microscope (SEM) photograph of embodiment 1.
Fig. 4 is the hydrogel material medicament slow release figure of embodiment 1.
Fig. 5 is the hydrogel material scanning electron microscope (SEM) photograph of embodiment 2.
Fig. 6 is the hydrogel material scanning electron microscope (SEM) photograph of embodiment 3.
Fig. 7 is the hydrogel material medicament slow release figure of embodiment 4.
Fig. 8 is the hydrogel material medicament slow release figure of embodiment 5.
Specific embodiment
Purposes, technical schemes and advantages in order to better illustrate the present invention, below in conjunction with the drawings and specific embodiments pairThe present invention is described further.
Test method employed in the embodiment of the present invention is conventional method unless otherwise specified；Used material,Reagent etc., is commercially available unless otherwise specified.
Embodiment 1
A kind of preparation method of polyvinyl alcohol hydrogel, includes the following steps:
(1) configure polyvinyl alcohol water solution: 10g polyvinyl alcohol is added in 100mL deionized water, stirred at 90 DEG C toDissolution obtains polyvinyl alcohol water solution, saves stand-by；
(2) preparation load winter Ling Su/sodium alginate micro ball compound: 1mL Tween 80 and 100mL olive oil are mixed,The sodium alginate aqueous solution that 20mL mass concentration is 2% is added drop-wise in olive oil by stirring at normal temperature 1h under 800r/min, is continued normalTemperature stirring 1h is eventually adding mass concentration and is stirred overnight for 2.5% calcium chloride solution and 50mg winter Ling Su；Solution isopropanolThree times with deionized water difference centrifuge washing, centrifugal speed 10000r/min, each centrifugation time is 15min, -60 DEG C lastFreeze-drying for 24 hours, obtains load winter Ling Su/sodium alginate micro ball compound；Fig. 1 is sodium alginate micro ball scanning electron microscope (SEM) photograph, byFig. 1 can be used for carrying medicament it is found that preparing resulting sodium alginate micro ball compound diameter is about 40 μm；
(3) it prepares polyethylenimine derivates carrier: 0.5g hyaluronic acid is dissolved in 100mL deionized water, be added0.2g EDC, 0.4g NHS, 2g polyethyleneimine, stirring at normal temperature is overnight, is dialysed with the bag filter that molecular cut off is 10KThree days, change water daily twice, be finally freeze-dried it is spare, freeze-drying condition be -60 DEG C freeze-drying for 24 hours；Fig. 2 is polyethyleneImine derivative carrier core magnetic chart, as shown in Figure 2, product have the spy of polyethyleneimine (PEI) and hyaluronic acid (HA) simultaneouslyLevy peak, therefore the provable polyethylenimine derivates carrier successfully synthesized for loading Microrna；
(4) preparation load mir-29A/ polyethylenimine derivates carrier: the mir-29A for being 20 μm of ol/L by 5 μ L concentrationSolution is added drop-wise in the polyethylenimine derivates carrier of 1mL step (3), obtains load mir-29A/ polyethylenimine derivatesCarrier, freezen protective at -20 DEG C；
(5) it prepares hydrogel material: step (1), step (2), step (4) obtained component respectively being taken into 1mL, mixed under room temperatureUniformly, it pours into mold, is placed in refrigerated overnight in -20 DEG C of refrigerators, is prepared into polyvinyl alcohol hydrogel.
Fig. 3 is the hydrogel material scanning electron microscope (SEM) photograph of the present embodiment, from the figure 3, it may be seen that hydrogel made from the present embodiment isSmooth porous structure belongs to typical hydrogel structure.
Medicament slow release test is carried out to hydrogel made from the present embodiment, Fig. 4 is hydrogel material medicament slow release figure, by scheming4 it is found that drug is in preceding 10 hours quick releases, and sustained release rate about 50%, subsequent rate slows down, until after 120 hours, medicament slow release rateUp to maximum value 90% or so.
Hydrogel made from the present embodiment is used for skin wound reparation, as it can be seen from table 1 control group is (without smallRNA hydrogel and experimental group (hydrogel material that the present embodiment contains Microrna)), therapeutic effect is deposited in the same timeAfter different, 14 days, the wound diameter of experimental group is about the half of control group, therefore, it can be seen that water-setting of the inventionGlue has gene therapy effect.
Table 1
| Time | Control group | Experimental group |
| 1st day | 10mm | 10mm |
| 3rd day | 8mm | 7mm |
| 7th day | 6mm | 4mm |
| 14th day | 3mm | 1.5mm |
Embodiment 2
A kind of preparation method of polyvinyl alcohol hydrogel material, includes the following steps:
(1) configure polyvinyl alcohol water solution: 15g polyvinyl alcohol is added in 100mL deionized water, stirred at 90 DEG C toDissolution obtains polyvinyl alcohol water solution, saves stand-by；
(2) preparation load winter Ling Su/sodium alginate micro ball compound: 1mL Tween 80 and 100mL olive oil are mixed,The sodium alginate aqueous solution that 20mL mass concentration is 2% is added drop-wise in olive oil by stirring at normal temperature 1h under 800r/min, is continued normalTemperature stirring 1h is eventually adding mass concentration and is stirred overnight for 2.5% calcium chloride solution and 100mg winter Ling Su；Solution isopropylThree times, centrifugal speed 10000r/min, each centrifugation time is 15min to pure and mild deionized water difference centrifuge washing, last -60DEG C freeze-drying for 24 hours, obtain load winter Ling Su/sodium alginate micro ball compound；
(3) it prepares polyethylenimine derivates carrier: 1g hyaluronic acid is dissolved in 100mL deionized water, be added0.4g EDC, 0.8g NHS, 4g aziridine, stirring at normal temperature is overnight, carries out dialysis three with the bag filter that molecular cut off is 10KIt, changes water two to three times daily, be finally freeze-dried it is spare, freeze-drying condition be -60 DEG C of freeze-dryings for 24 hours；
(4) preparation load mir-29A/ polyethylenimine derivates carrier: the mir-29A for being 20 μm of ol/L by 5 μ L concentrationSolution is added drop-wise in the polyethylenimine derivates carrier of 1mL step (3), obtains load mir-29A/ polyethylenimine derivatesCarrier, freezen protective at -20 DEG C；
(5) it prepares hydrogel material: step (1), step (2), step (4) obtained component respectively being taken into 1mL, mixed under room temperatureUniformly, it pours into mold, is placed in refrigerated overnight in -20 DEG C of refrigerators, polyvinyl alcohol hydrogel is prepared.
Fig. 5 is the hydrogel material scanning electron microscope (SEM) photograph of the present embodiment, and as shown in Figure 5, hydrogel made from the present embodiment isSmooth porous structure belongs to typical hydrogel structure.
Embodiment 3
A kind of preparation method of polyvinyl alcohol hydrogel material, includes the following steps:
(1) configure polyvinyl alcohol water solution: 20g polyvinyl alcohol is added in 100mL deionized water, stirred at 90 DEG C toDissolution obtains polyvinyl alcohol water solution, saves stand-by；
(2) preparation load winter Ling Su/sodium alginate micro ball compound: 1mL Tween 80 and 100mL olive oil are mixed,The sodium alginate aqueous solution that 20mL mass concentration is 3% is added drop-wise in olive oil by stirring at normal temperature 1h under 800r/min, is continued normalTemperature stirring 1h is eventually adding mass concentration and is stirred overnight for 5% calcium chloride solution and 100mg winter Ling Su；Solution isopropanolThree times with deionized water difference centrifuge washing, centrifugal speed 10000r/min, each centrifugation time is 15min, -60 DEG C lastFreeze-drying for 24 hours, obtains load winter Ling Su/sodium alginate micro ball compound；
(3) it prepares polyethylenimine derivates carrier: 1g hyaluronic acid is dissolved in 100mL deionized water, be added0.4g EDC, 0.8g NHS, 4g polyethyleneimine, stirring at normal temperature is overnight, is dialysed with the bag filter that molecular cut off is 10KThree days, change water two daily to three times, be finally freeze-dried it is spare, freeze-drying condition be -60 DEG C of freeze-dryings for 24 hours；
(4) preparation load mir-29A/ polyethylenimine derivates carrier: the mir-29A for being 20 μm of ol/L by 8 μ L concentrationSolution is added drop-wise in the polyethylenimine derivates carrier of 1mL step (3), obtains load mir-29A/ polyethylenimine derivatesCarrier, freezen protective at -20 DEG C；
(5) it prepares hydrogel material: step (1), step (2), step (4) obtained component respectively being taken into 1mL, mixed under room temperatureUniformly, it pours into mold, is placed in refrigerated overnight in -20 DEG C of refrigerators, polyvinyl alcohol hydrogel is prepared.
Fig. 6 is the hydrogel material scanning electron microscope (SEM) photograph of the present embodiment, it will be appreciated from fig. 6 that hydrogel made from the present embodiment isSmooth porous structure belongs to typical hydrogel structure.
Embodiment 4
A kind of preparation method of polyvinyl alcohol hydrogel material, includes the following steps:
(1) it configures polyvinyl alcohol water solution: stirring and dissolving in deionized water is added in polyvinyl alcohol, obtains polyvinyl alcohol waterSolution, the mass concentration of polyvinyl alcohol water solution are 5%, and solution temperature is 80 DEG C, mixing speed 300r/min；
(2) winter Ling Su preparation load winter Ling Su/sodium alginate micro ball compound: is added to sodium alginate micro ball solutionIn, freeze-dried back；Sodium alginate micro ball the preparation method comprises the following steps: Tween 80 and olive oil are mixed to obtain mixed liquor, room temperatureStirring, sodium alginate aqueous solution is added drop-wise in mixed liquor, continues stirring at normal temperature, and calcium chloride solution is added and is stirred overnight, thenThree times with isopropanol and deionized water difference centrifuge washing, freeze-drying obtains sodium alginate micro ball；Sodium alginate micro ball solutionConcentration be 5mg/mL, winter Ling Su 0.5mg is added in every milliliter of sodium alginate micro ball solution, and freeze-drying temperature is -60 DEG C, freezingDrying time is for 24 hours；
(3) prepare polyethylenimine derivates carrier: using deionized water as solvent, configuration quality score be 0.5% it is saturating0.1g 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide, 0.2g N- hydroxyl is added in bright matter acid solution, every gram of hyaluronic acidSuccinimide, 1g polyethyleneimine, stirring at normal temperature is overnight, carries out dialysis three days with the bag filter that molecular cut off is 10K, oftenIt is changed water 2 times, be finally freeze-dried it is spare, freeze-drying temperature be -60 DEG C, sublimation drying be for 24 hours, obtain polyethyleneimineDerivative carrier；
(4) miR-29A solution preparation load miR-29A/ polyethylenimine derivates carrier: is added drop-wise to step (3) instituteIn the polyethylenimine derivates carrier obtained, freezen protective；The dosage of miR-29A is every milligram of polyethylenimine derivates carrierMiddle addition 50ng miR-29A；
(5) it prepares polyvinyl alcohol hydrogel: step (1), step (2), step (4) obtained component is mixed in equal volume, fillEnter in mold, is placed in refrigerated overnight at -20 DEG C, obtains polyvinyl alcohol hydrogel.
Medicament slow release test is carried out to hydrogel made from the present embodiment, Fig. 7 is hydrogel material medicament slow release figure, by scheming7 it is found that drug is in preceding 10 hours quick releases, and sustained release rate about 60%, subsequent rate slows down, until after 80 hours, medicament slow release rateUp to maximum value 90% or so.
Hydrogel made from the present embodiment is used for skin wound reparation, from table 2 it can be seen that control group (embodiment 3)Hydrogel and experimental group (the present embodiment), there is some difference for therapeutic effect in the same time, after 14 days, the wound of control groupMouth diameter is about the half of experimental group, therefore, it can be seen that the decline that the winter insults cellulose content is unfavorable for wound reparation, but the present embodimentStill there is certain therapeutic effect.
Table 2
Embodiment 5
A kind of preparation method of polyvinyl alcohol hydrogel material, includes the following steps:
(1) it configures polyvinyl alcohol water solution: stirring and dissolving in deionized water is added in polyvinyl alcohol, obtains polyvinyl alcohol waterSolution, the mass concentration of polyvinyl alcohol water solution are 20%, and solution temperature is 90 DEG C, mixing speed 500r/min；
(2) winter Ling Su preparation load winter Ling Su/sodium alginate micro ball compound: is added to sodium alginate micro ball solutionIn, freeze-dried back；Sodium alginate micro ball the preparation method comprises the following steps: Tween 80 and olive oil are mixed to obtain mixed liquor, room temperatureStirring, sodium alginate aqueous solution is added drop-wise in mixed liquor, continues stirring at normal temperature, and calcium chloride solution is added and is stirred overnight, thenThree times with isopropanol and deionized water difference centrifuge washing, freeze-drying obtains sodium alginate micro ball；Sodium alginate micro ball solutionConcentration be 10mg/mL, winter Ling Su 2mg is added in every milliliter of sodium alginate micro ball solution, and freeze-drying temperature is -60 DEG C, freezingDrying time is for 24 hours；
(3) prepare polyethylenimine derivates carrier: using deionized water as solvent, configuration quality score be 2% it is transparent0.5g 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide, 1g N- hydroxysuccinimidyl is added in matter acid solution, every gram of hyaluronic acidAcid imide, 5g polyethyleneimine, stirring at normal temperature is overnight, carries out dialysis three days with the bag filter that molecular cut off is 10K, changes dailyWater 3 times, finally it is freeze-dried spare, freeze-drying temperature is -60 DEG C, and sublimation drying is for 24 hours, it is derivative to obtain polyethyleneimineObject carrier；
(4) miR-29A solution preparation load miR-29A/ polyethylenimine derivates carrier: is added drop-wise to step (3) instituteIn the polyethylenimine derivates carrier obtained, freezen protective；The dosage of miR-29A is every milligram of polyethylenimine derivates carrierMiddle addition 100ng miR-29A；
(5) it prepares polyvinyl alcohol hydrogel: step (1), step (2), step (4) obtained component is mixed in equal volume, fillEnter in mold, is placed in refrigerated overnight at -20 DEG C, obtains polyvinyl alcohol hydrogel.
Medicament slow release test is carried out to hydrogel made from the present embodiment, Fig. 8 is hydrogel material medicament slow release figure, by scheming8 it is found that drug is in preceding 10 hours quick releases, and sustained release rate about 50%, subsequent rate slows down, until after 100 hours, medicament slow release rateUp to maximum value 90% or so.
Hydrogel made from the present embodiment is used for skin wound reparation, from table 3 it can be seen that control group (embodiment 3)Hydrogel and experimental group (the present embodiment), therapeutic effect is identical in the same time, it can be seen that miR-29A content is increased toUnobvious to wound repair raising after to a certain degree, experimental group and control group all have preferable therapeutic effect.
Table 3
| Time | Control group | Experimental group |
| 1st day | 10mm | 10mm |
| 3rd day | 7mm | 7mm |
| 7th day | 4mm | 4mm |
| 14th day | 1.5mm | 1.5mm |
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present inventionThe limitation of range is protected, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art shouldUnderstand, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the essence of technical solution of the present inventionAnd range.