CN103536960B - Hydrogel capable of slowly releasing antibacterial peptide as well as preparation method thereof - Google Patents
Hydrogel capable of slowly releasing antibacterial peptide as well as preparation method thereof Download PDFInfo
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- CN103536960B CN103536960B CN201310520058.4A CN201310520058A CN103536960B CN 103536960 B CN103536960 B CN 103536960B CN 201310520058 A CN201310520058 A CN 201310520058A CN 103536960 B CN103536960 B CN 103536960B
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- antibacterial peptide
- hyaluronic acid
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- 239000000017 hydrogel Substances 0.000 title claims abstract description 32
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- 108700042778 Antimicrobial Peptides Proteins 0.000 claims description 20
- 102000044503 Antimicrobial Peptides Human genes 0.000 claims description 20
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims description 18
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Abstract
The invention relates to a hydrogel capable of slowly releasing an antibacterial peptide as well as a preparation method thereof. Medical hydrogel dressing with better biocompatibility and medicine safety is prepared by embedding anthropogenic antibacterial peptide molecules by using glycosaminoglycan molecules through in situ polymerization; a hydrogel antibacterial peptide medicine slow release system is formed by controlling the concentration of the embedded anthropogenic antibacterial peptide molecules and the number of hydrazide derivatives and aldehyde derivatives of glycosaminoglycan. The hydrogel provided by the invention has the characteristics of greenness and environmental friendliness, simplicity and convenience in operation and simplicity in process. The prepared glycosaminoglycan-based hydrogel can slowly release carrier materials and has an ability of controlled release of behaviors of the antibacterial peptide.
Description
Technical field:
The present invention relates to a kind of hydrogel and preparation method thereof that can be used for medical trauma nursing, particularly relate to a kind of can the glycosaminoglycans based aquagel and preparation method thereof of sustained-release antimicrobial peptide.
Background technology:
Antibacterial peptide is that the one produced through induction in organism has bioactive micromolecule polypeptide, and molecular weight, at about 2000 ~ 7000Da, is made up of 20 ~ 60 amino acid residues.Microbial antibacterial peptide, Antimicrobial Peptides From Plants, Antibacterial Peptide From Animals and human antibiotic peptide etc. are divided into by antibacterial peptide source.Antibacterial peptide obtains extensive research in repair in trauma field in recent years, bibliographical information people derived antimicrobial peptide (Host Defense Peptide) has broad-spectrum antiseptic, participation wound inflammation regulates, short impingement angiogenesis, the function of short impingement epithelium regeneration.In this complex physiologic process of human body wound healing, antibacterial peptide, as one of immune key signal molecule of interior raw type, interacts with various types of cells and various somatomedin, coordinates to reach poised state, realizes repair in trauma.Antibacterial peptide is in inducible expression in body, and during normal physiological condition, concentration is only 0.4 ~ 1 μm of ol/L.Be subject to up-regulated when alien bacteria, inflammation or wound stimulate, can detect that at scytitis place people's derived antimicrobial peptide concentration is high to 10 μm of ol/L.
Glycosaminoglycans (glycosaminoglycans, GAG) be by repeat disaccharide unit form without branch long chain polysaccharides.Its disaccharide unit is made up of aminohexose (glucosamine or aminogalactose) and alduronic acid usually, but in keratan sulfate, alduronic acid is replaced by galactose.Aminoglycan can be divided into six kinds, that is: hyaluronic acid, chondroitin sulfate, dermatan sulfate, Heparan sulfate, heparin, keratan sulfate according to the difference of composition glycosyl, connected mode, degree and position.Glycosaminoglycans and Fibrilla collagen, hyaluronic acid, Dan Baiduotang proteoglycan PG and glycoprotein form extracellular matrix (Extracellular Matrix, ECM) in varing proportions.Be distributed widely in the various tissue of animal and human's body, the physical action such as performance support, connection, water conservation, protection that extracellular matrix is not only static, and dynamically comprehensive impact produced on cell.
Since the seventies in last century, manufacturer of American-European countries starts to develop non-cotton Medical dressing product processed, replaces the dressing of normal gauze binder class with this.The hydrogel medical dressing product utilizing non-toxic high molecular material to develop is a kind of high-end medical dressing product, there is good biocompatibility, do not affect the metabolic process of life entity, and metabolite is discharged by hydrogel, like this under moistening environment, wound can not be formed a scab, and faster than healing in dry environments.
Summary of the invention:
The present invention proposes the design and preparation method that people's derived antimicrobial peptide and hydrogel are combined, by the antibacterial peptide hydrogel using the method to prepare, there is the advantage of good biocompatibility and antimicrobial peptide medicaments slow release.
With the glycosaminoglycans macromolecular material of good biocompatibility for primary raw material, in-situ cross-linked reaction of the prior art is adopted to prepare novel hydrogels dressing substrate, by peptide molecule and glycosaminoglycans macromolecular material conjugation reaction, and then embed peptide molecule by in-situ polymerization, add thermal sensitivity biomaterial simultaneously, be mixed and made into hydrogel.In preparation process, glycosaminoglycans molecular material and the total content of thermal sensitivity biomaterial in described hydrogel are 1%-80% (w/w).The proportioning of biological polysaccharide polymer and antibacterial peptide molecule is (10:1 to 200:1), and the proportioning of biological polysaccharide polymer and thermal sensitivity biomaterial is (1:1 to 1:10).By controlling be embedded the hydrazides derivant of people's derived antimicrobial peptide molecular concentration and glycosaminoglycans and the proportioning formation hydrogel antimicrobial peptide medicaments slow-released system of aldehyde radical derivant.
Glycosaminoglycans includes but not limited to following material: hyaluronic acid, chondroitin sulfate, dermatan sulfate, Heparan sulfate, heparin, keratan sulfate.Temperature-sensitive material includes but not limited to following material: poly-third ethylene polyethylene and ethylene copolymers, polyoxypropylene polyethylene and ethylene copolymers.People's derived antimicrobial peptide includes but not limited to following kind: alexin (Defensins) family, cathelicidins (Cathelicidins) family, be rich in histidine protein (Histatins) family, and glycine-rich protein (Hglyrichin) family.
The present invention relates to the production process of people's derived antimicrobial peptide, production technology is: be to adopt in prior art chemosynthesis or adopt engineered method, use bioreactor that the carrier containing people's derived antimicrobial peptide gene is gone out people's derived antimicrobial peptide at cells, separation and purification can obtain.Reversed-phase high-performance liquid chromatography (rHPLC) has good separating effect, resolution is high, the response rate is high feature, prepares polypeptide, high performance liquid chromatography also can be adopted to carry out separation and purification through chemosynthesis.RHPLC method is used to measure for the release behavior of antibacterial peptide hydrogel.Have studied the impact of the parameters such as polymer concentration, drug loading, polymer composition and sol-gel for drug release behavior, to obtain optimal drug slow-released system.Drug release patterns present initial prominent release with certain hour after the feature of slow releasing.Higher polymer concentration causes sustained release rate to slow down, and early stage dashing forward releases reduction.This is because higher polymer concentration causes hydrogel structure more closely, thus make the diffusion of medicine slack-off.Similar, higher polymer molecular weight also makes medicament slow release slack-off.On the contrary, drug loading is little for the impact of drug release, and main cause is that medicine water solublity is fine.In all laboratory samples, the release rate of antibacterial peptide is close to 90%.
The present invention, also by optimizing hydrogel formulation, introducing the strong material of heat sensitivity (as poly-third ethylene polyethylene and ethylene copolymers and/or polyoxypropylene polyethylene and ethylene copolymers), making the physical state of hydrogel below 10 DEG C almost close to liquid.When the injection of cryopreservation is expelled to wound surface, aqueous water gel trickles rapidly to any tiny area of wound, and stop in 10 minutes body surface 37 DEG C, its physical property converts to close to solid phase, forms gel, covers wound.
The present invention mainly through using glycosaminoglycans molecule in-situ polymerization embedding people derived antimicrobial peptide molecule, prepares a kind of medical hydrogel dressings with good biocompatibility and drug safety.The antibacterial peptide hydrogel utilizing glycosaminoglycans macromolecular material to develop is when tissue repair, both the passage of seed cell propagation and the place adhered to and metabolism had been provided, again for the growth of cell provides nutrition, and there is the antibacterial effect of antiinflammatory, avoid the hypertrophy phenomenon in tissue repair and wound healing process, provide favourable environment for wound better heals, avoid the generation of cicatrix.
Marginal data:
In order to deepen the understanding of the present invention, below in conjunction with embodiment and accompanying drawing, the invention will be further described.But be not limitation of the invention further, foregoing according to the present invention is made other forms of change, replacement etc. and is all belonged to scope of the present invention.Wherein:
Fig. 1: the release in vitro kinetic curve after antibacterial peptide parcel.
Fig. 2: the external degradation of the hyaluronic acid after uncrosslinked and crosslinked under hyaluronidase effect.
Detailed description of the invention:
One, the preparation of people's derived antimicrobial peptide and separating-purifying
1, purification experimental procedure
Employing solid-state chemical reaction method method synthesizes the LL-37 in cathelicidins (cathelicidins) family, aminoacid sequence LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES, improvement on synthesis crude product is used after dissolving containing acetonitrile solution, centrifugal, retain supernatant, discard precipitation.Reversed-phase liquid chromatography system is, SCL210AVP chromatograph of liquid, comprises system controller, LC210ATVP chromatogram pump, injection valve, SPD2M10AVP diode array detector, CLASS2VP5.33 chromatographic work station.Chromatographic column is the stainless steel tube of 200 × 4mm I.D., with homogenate method filling reversed phase chromatography filler.Get certain density polypeptide sample solution sample introduction on the reversed-phase high-performance liquid chromatography (rHPLC) having used mobile phase A liquid [water+0.1%TFA (VPVP%)] equilibrated, then linear gradient elution is carried out until meet the requirements of B liquid concentration [acetonitrile+0.1%TFA (VPVP%)], the LL-37 purity that separation and purification obtains is greater than 95%, collects obtain white powder through lyophilizing.
2, antibacterial activity evaluation
Test the activity to Grain-positive and gram-negative bacteria of LL-37 antibacterial peptide after purification respectively, the escherichia coli and the golden yellow staphylococcus of multidrug resistance that comprise resistance to ampicillin and streptomycin are as shown in table 1.Testing result shows, has antibacterial activity to gram-positive bacteria (Acinetobacter baumannii, anthrax bacillus and golden yellow staphylococcus) and gram-negative bacteria (escherichia coli).And hemolytic result of the test shows, LL-37 has the characteristic of low hemolytic.
Half-inhibition concentration and the hemolytic of table 1LL-37 antibacterial activity detect
Two, the conjugation reaction of polysaccharide and polypeptide
1, covalent bonding antibacterial peptide
By two-step reaction, hyaluronic acid realizes aldehyde radical under the Oxidation of periodic acid, and the amino in the hyaluronic acid of aldehyde radical and LL-37 peptide carries out conjugation condensation and forms aldehyde radical derivant.Sodium metaperiodate 0.1M, DHA HA (molecular weight 500kD, concentration 2%) realize aldehyde radical, make it have multiple aldehyde radical, under pH=8.0 condition, the hyaluronic acid (HA-ADH) of aldehyde radical makes to react completely with LL-37 peptide conjugation reaction for 4 hours, and conjugation percentage ratio is 10%.Conjugation Percentage definitions is: tested by trinitro-benzene-sulfonic acid and sample at 334nm place absorb, the ratio of aldehyde radical quantity in polymer molecule before and after reaction.
2, gel is prepared
Synthesized gel rubber technology of preparing, with hyaluronic acid and polyvinyl alcohol for adjuvants such as primary raw material and poly-third ethylene polyethylene and ethylene copolymers, adopts in-situ cross-linked technology to prepare aerogel dressing substrate, embeds people's derived antimicrobial peptide molecule by in-situ polymerization.Concrete scheme: in the solution after previous step reaction terminates, add by 1:1 by the mol ratio of HA-peptide aldehyde radical derivant and hyaluronic acid hydrazides derivant (HA), stirring reaction 6 hours at 4 DEG C, period adds heat sensitizer polyoxypropylene polyethylene (1:2 mol ratio).The HA (HA-ADH) of hydrazide structure can be synthesized with adipyl dihydrazide (ALD) dichloroethane solution and I-hydroxybenzotriazole (HOBt) by hyaluronic acid HA in advance under pH6.8 condition.
Three, the degradability of antibacterial peptide hydrogel and medicament slow release ability
1, external medicine slow-release capability
The hydrogel of 1 gram of parcel antibacterial peptide is added 40mlPBS solution and is placed on 37 DEG C.Took out 1ml aqueous solution in water bath with thermostatic control to add the new PBS solution of 1ml take out LL-37 concentration in solution and use rHPLC method to carry out continuous 14 days mensuration (Fig. 1) for the release behavior of antibacterial peptide hydrogel to keep constant mensuration of 100mlPBS liquor capacity every 24 hours.Display release antibacterial peptide molecule speed is 300 micromoles per liter/sky, can be not less than 14 days by continuous release.
2, degradability evaluation
Hyaluronidase (hyaluronidase, HAase) is that one can be hydrolyzed hyaluronic enzyme.Antibacterial peptide hydrogel degradability is also embodied in, 1g gel is added in the phosphate buffer containing 33U hyaluronidase, 37 DEG C effect 48 hours after, use the dextran of known molecular amount to make standard substance to detect hyaluronic molecular weight (Fig. 2), the enzymatic degradation rate being bonded with the cross-linked hyaluronic acid gel of antibacterial peptide is starkly lower than without crosslinked hyaluronic acid.
Four, Wound care applicating evaluating
12 rats, about 200 grams, anesthesia back part shaves hair, and draw 5 millimeters deep with scalpel, 3 centimeter length wounds, are divided into two groups at random, often organizes 6, and wherein one group of wound applies by about 1cc antibacterial peptide hydrogel every day, and matched group uses normal saline.The physical state of gel injection below 10 DEG C is almost close to liquid, when the injection of cryopreservation is expelled to wound surface, carrying antibacterial peptide fraction trickles rapidly to any tiny area of wound, stop in 5 to 10 minutes body surface 37 DEG C, its physical property converts to close to solid phase, form gel, cover wound.After results of animal display uses antimicrobial peptide LL-37 aerogel dressing fortnight, statistical result shows that rat back wound healing is significantly better than matched group.
Claims (3)
1. can the preparation method of sustained-release antimicrobial peptide hydrogel, its step is as follows:
(1) preparation of antibacterial peptide: the polypeptide crude product adopting solid-state chemical reaction method method synthetic antibacterial peptide LL-37, by the polypeptide crude product of synthesis with containing after acetonitrile solution dissolving, centrifugal, retain supernatant, discard precipitation, obtain antimicrobial peptide LL-37;
(2) covalent bonding antibacterial peptide: the amino in the hyaluronic acid of aldehyde radical and antimicrobial peptide LL-37 peptide carries out conjugation condensation and forms hyaluronic acid-peptide aldehyde radical derivant;
Reaction condition is: pH=8.0, and the response time is: 4 hours;
The hyaluronic acid of described aldehyde radical is prepared by sodium metaperiodate 0.1M and DHA;
The molecular weight of described DHA HA is 500kD, and concentration is 2%;
(3) preparation of gel:
Aerogel dressing substrate is prepared by in-situ cross-linked technology, hyaluronic acid step (2) obtained-peptide aldehyde radical derivant and hyaluronic acid hydrazides derivant in molar ratio 1:1 add in hydrogel matrix after mixing, stirring reaction 6 hours at 4 DEG C, period adds polyoxypropylene polyethylene, and can obtain can the antibacterial peptide hydrogel of slow release;
The preparation method of described hyaluronic acid hydrazides derivant is: synthesized under pH=6.8 condition by the dichloroethane solution of hyaluronic acid and adipyl dihydrazide and I-hydroxybenzotriazole;
In described polyoxypropylene polyethylene, the mol ratio of polyoxypropylene and polyethylene segment is 1:2;
Described the antibacterial peptide hydrogel of slow release can have antimicrobial peptide medicaments slow-released system, and release antibacterial peptide molecule speed is 1 ~ 300 micromoles per liter/sky, can be not less than 14 days by continuous release.
2. can a sustained-release antimicrobial peptide hydrogel, it is characterized in that: prepared by method according to claim 1.
3. an antimicrobial peptide medicaments slow-released system, is characterized in that: be prepared by method according to claim 1, and is produced by control the to be embedded molecular concentration of LL-37 and the proportioning of hyaluronic hydrazides derivant and aldehyde radical derivant.
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