CN102198081B - Leptin gel slow-release system and application thereof - Google Patents
Leptin gel slow-release system and application thereof Download PDFInfo
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- CN102198081B CN102198081B CN2011101303510A CN201110130351A CN102198081B CN 102198081 B CN102198081 B CN 102198081B CN 2011101303510 A CN2011101303510 A CN 2011101303510A CN 201110130351 A CN201110130351 A CN 201110130351A CN 102198081 B CN102198081 B CN 102198081B
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- polyethylene glycol
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Abstract
The invention discloses a preparation method and application of a leptin-chitosan-monomethoxypolyethylene glycol gel administration system. The gel system comprises the following components in percentage by weight: 3.5% of chitosan, 3.0% of monomethoxypolyethylene glycol and collagen. The leptin gel administration system disclosed by the invention has good temperature sensibility and mild preparation conditions, reduces the leptin degradation rate and can be used for intra-articular injection.
Description
Technical field
The present invention relates to a kind of gel rubber sustained-release system of medicine.Particularly, the invention discloses the method for preparing and the application thereof of the chitosan-mono methoxy polyethylene glycol gel delivery system of leptin.
Background technology
Chitosan (chitosan) is a kind of acetyl derivative that takes off of chitin, is a large amount of alkaline polysaccharides that exist of nature, and bio-toxicity is little; Have the favorable tissue compatibility, biodegradability and adhesion, obtained deep research and used [Shi X W, Du Y M widely in medical science, field of biology; Sun L P; Et al.Macromolecular Bioscience, 2005,5:881889].Chitosan also is a kind of ideal medical embedded material [Khor E, Lim L Y.Biomaterials, 2003,24:23392349].But in the ordinary course of things, chitosan only is dissolved in the acidic aqueous solution, in neutrality or alkaline solution, can form the deposition of similar gels, and this process and temperature relation are little.But Recent study finds, through adding basic salt, hydroxyl polymer-containing or chitosan being carried out derivatization and graft reaction, can obtain having the chitosan-based physical gel of temperature sensitivity.Solution keeps solution state under physiological pH (7.0) and room temperature, and rapid gelation when temperature rises to body temperature (37 ℃).
(Methoxy Polyethylene Glycol mPEG) has good water-solubility, wettability, lubricity, physiology inertia to mono methoxy polyethylene glycol, and is non-stimulated, gentle to human body, in cosmetics and pharmaceuticals industry, is widely used, and molecular weight is 2KD.In the acid solution of chitosan, add an amount of mono methoxy polyethylene glycol and with the proton of chitosan chitosan is precipitated in can be not at once, can make the pH value of chitosan solution rise to physiological range be about pH7.0.Under cryogenic conditions, the effect of chitosan and water has stoped the gathering of chitosan chain; When temperature raise, the structuring effect of mono methoxy polyethylene glycol and water (this interaction energy weakens the effect of chitosan and water) was strengthened, and made the dehydration of chitosan chain, and the zone of hydrone is replaced by the hydroxyl group of Polyethylene Glycol, the chitosan chain aggregation.
The biological activity of chitosan and PEG and biocompatibility are very clear and definite, aspect drug-supplying system, important use are arranged, and existing a lot of work combine the carrier as medicine with chitosan and PEG.[Bhattarai N, Ramay H R, Gunn J such as Bhattarai; Et al.Journal of Controlled Release; 2005,103:609 624] with the situ-gel material of these two kinds of materials combination preparations, as model drug the release in vitro function of this gel is investigated with BSA; Drug release reached more than 40 days, and the structure of BSA is not seen change in the dispose procedure.
1994; People such as the Zhang [Zhang of Rockefeller university; Et al.Positional cloning of the mouse obese gene and it ' s human homologue.Nature; 1994,372:425～426] utilize molecular biology method successfully to clone mice and people's ob gene.This gene is mainly expressed in the white adipose cell, product be a kind of by 167 amino acid residues form, molecular weight 16000 daltonian proteohormones.Because of making animal, this material reduces, so be named as " leptin (Leptin) "." Leptin " comes from Greece's word " Leptos " and means " thin ".Obese type (ob/ob) mice is because its ob gene in the sudden change of the 105th generation by C → T, makes the arginine codon CGA in this site change termination codon TGA into.This sudden change makes ob gene expression product loss of activity, has not promptly had leptin secretion, so the obesity of showing as.
Leptin in the blood circulation is 146 amino acid whose peptide hormones, and its active site contains 146 amino acid residues, and relative molecular mass is 16KD, and hydrophilic is strong, is present in the blood plasma with monomeric form.Similar with the secretion of other hormones, the secretion of leptin also is pulsed.Leptin mRNA expresses and is the diurnal periodicity variation, and night, expression was the highest, and the half-life is that its secretion of 9.4 ± 3.0min has certain rhythmicity.Leptin is a kind of many target organs, a function protein hormones widely, and its major physiological function comprises: suppress to ingest, increase energy expenditure; Regulate growth promoter; Short epithelial cell, angiogenic growth; Influence bone reconstruction etc.
Leptin has and reduces animal appetite, improves energy metabolism efficient, increases energy consumption, reduces the fat storage, and effect such as lose weight.People such as Haalas [Haalas; Et al.Weight-reduction effects of the plasma protein encoded by the obese gene.Science; 1995,269:543～546] give ob/ob injected in mice gene recombinaton leptin, its body weight significantly descends.Inject leptin to mouse peritoneal every day, and the feed intake of ob/ob mice descends than matched group behind the 4d, and weight loss reaches 40% after 60%, 4 week.Simultaneously, the activity of mice increase, metabolism reinforcement, plasma insulin and blood sugar level reduce.
People such as Khalid [Khalid; Et al.Leptin treatment rescues t he sterility of genetically chese ob/ob males.Endocrinology; 1997,138:1190～1193] report, the ob/ob mice is except obesity; Also have a marked feature, i.e. genital atrophy and sterile throughout one's life.The injection of exogenous leptin can make the puberty of rat shift to an earlier date, and shows that leptin plays an important role to the puberty startup of animal.
But leptin stimulation in rats bone marrow stem cell propagation is with the propagation of the collaborative hemopoietic cell of granulocyte stimulating factor.Angiogenesis factor synergism such as leptin and VEGF and fibroblast growth factor promote angiogenesis in addition.
Leptin can significantly improve level and the proteinic secretion thereof of bone protected protein (OPG) mRNA; Reduce the level of nucleus factor K B receptor activation factor aglucon (RANKL) mRNA; Thereby the activation, the maturation that suppress osteoclast; The bone of prompting leptin scalable rat is rebuild, and helps bone formation, prevents the bone loss that removal ovary causes.
For improving the leptin half-life in vivo; The present invention is according to the physicochemical property of chitosan and mono methoxy polyethylene glycol; Optimize the condition that in the past prepares chitosan and mono methoxy polyethylene glycol hydrogel system, successfully prepared the high performance Atrigel of leptin.
Summary of the invention
The invention discloses a kind of gel delivery system of leptin, containing concentration is 10
-5-10
-4The chitosan of the leptin of M, percentage by weight 3.0%-3.5%, 3.0%-3.5% mono methoxy polyethylene glycol and 0.01%-0.1% collagen protein.
The present invention has confirmed chitosan and mono methoxy polyethylene glycol mixed proportion, is according to the chitosan of the 3%-3.5% of weight ratio, the mono methoxy polyethylene glycol of 3.0%-4.5%.
The invention also discloses a kind of method for preparing of temperature sensitive leptin aquagel, may further comprise the steps:
1) preparation of mono methoxy polyethylene glycol solution: take by weighing mono methoxy polyethylene glycol, be dissolved in distilled water, sustained oscillation on the oscillating agitator makes its thorough dissolving, and processing concentration is 30% mono methoxy polyethylene glycol solution (w/v), membrane filtration, and low temperature storage is subsequent use.
2) preparation of chitosan solution: use acetic acid preparing chitosan solution, stirs after 2 days behind the chitosan solution autoclave sterilization with acquisition, use after being cooled to room temperature.
3) preparation of leptin-collagen solution: collagen is dissolved in the sodium hydroxide, and regulates pH value, add leptin again and make its dissolving to 7.0-7.4.
4) preparation of leptin-chitosan-mono methoxy polyethylene glycol gel: behind three kinds of solution ice bath 30min; Draw chitosan-acetic acid solution in container; After dripping leptin-collagen solution, stirring dropwise adds mono methoxy polyethylene glycol solution simultaneously fast, and continues to stir 20min.In the leptin-chitosan-mono methoxy polyethylene glycol gel systems that obtains, the concentration of leptin is 10
-5-10
-4M, chitosan mass concentration are 3.0%-3.5%, and mono methoxy polyethylene glycol concentration is 3.0%-3.5%, 0.01%-0.1% collagen protein.
The leptin gel delivery system that the present invention developed was with document and patent document had more technological accuracy in the past.In chitosan and mono methoxy polyethylene glycol hybrid system, the chitosan of 2% following concentration is difficult to solidify under physiological temp, and the concentration above 4.0% then is difficult to dissolving; The present invention also confirmed simultaneously under the chitosan of the 3.0%-3.5% of weight ratio, in the temperature-sensitive hydrogel system, and the accurate concentration of mono methoxy polyethylene glycol.
Leptin gel delivery of the present invention system, long half time helps the comparatively secular effectiveness of leptin performance.The invention also discloses the purposes of leptin gel delivery system, comprise that joint cavity injection leptin gel delivery system is with treatment bone loss and bone healing.
Description of drawings
The SDS-PAGE electrophoretogram of Fig. 1 leptin.Wherein swimming lane 1 is a leptin protein.
The gel time of the chitosan of Fig. 2 variable concentrations and mono methoxy polyethylene glycol combination.1:3.5% chitosan and 4.0%; 2:3% chitosan and 4.0%; 3:3.5% chitosan and 3.0%; 4:3.0% chitosan and 3.0%.
Fig. 3 leptin compares in release in vitro at chitosan-mono methoxy polyethylene glycol hydrogel system and at phosphate buffer.
The preparation of embodiment one leptin gel rubber sustained-release drug-supplying system
Experiment material
Chitosan (Promega; The U.S.), mono methoxy polyethylene glycol (Sigma; The U.S.), leptin (laboratory self-control), glacial acetic acid (Beijing chemical reagents corporation), acetonitrile (Beijing chemical reagents corporation), sodium hydroxide (Beijing chemical reagents corporation), the two third rare amide (Sigma, the U.S.) of third rare amide & methene, sodium hydrogen phosphate (Beijing chemical reagents corporation), sodium dihydrogen phosphate (Beijing chemical reagents corporation).
Experimental technique
1) protein expression of leptin and purification
1. get people's fatty tissue and extract total RNA, reverse transcription becomes cDNA, with leptin upstream and downstream primer Sequnence NO.1, the Sequnence NO.2 amplifying target genes of design, has obtained the full-length gene of leptin, and its sequence is Sequnence NO.3.
2. complete synthesis leptin gene is connected on the expression vector, and is transformed in the expression bacterium.
3. the IPTG that adds final concentration and be 0.1mM induces the expression of leptin gene.
4. with the cracking of bacterium liquid, through nickel ion affinity column purification leptin protein.
2) preparation of leptin-chitosan-mono methoxy polyethylene glycol gel delivery system
1. Determination of deacetylating degree of chitosan: measure with elemental analyser, adopt CHN S pattern, measure the constituent content of material respectively with CHN, the deacetylation of calculating chitosan, the result shows that its deacetylation is 90%.
2. the preparation of mono methoxy polyethylene glycol solution: take by weighing mono methoxy polyethylene glycol, be dissolved in distilled water, sustained oscillation on the oscillating agitator makes its thorough dissolving, and processing concentration is 30% mono methoxy polyethylene glycol solution (w/v), membrane filtration, and low temperature storage is subsequent use.
3. the preparation of chitosan solution: use acetic acid preparing chitosan solution, stirs after 2 days behind the chitosan solution autoclave sterilization with acquisition, use after being cooled to room temperature.
4. the preparation of leptin-collagen solution: collagen is dissolved in the sodium hydroxide, and regulates pH value, add leptin again and make its dissolving to 7.0-7.4.
5. the preparation of leptin-chitosan-mono methoxy polyethylene glycol gel: behind three kinds of solution ice bath 30min; Draw chitosan-acetic acid solution in container; After dripping leptin-collagen solution, stirring dropwise adds mono methoxy polyethylene glycol solution simultaneously fast, and continues to stir 20min.In the leptin-chitosan-mono methoxy polyethylene glycol gel systems that obtains, the concentration of leptin is 10
-5-10
-4M, chitosan mass concentration are 3.0%-3.5%, and mono methoxy polyethylene glycol concentration is 3.0%-3.5%, 0.01%-0.1% collagen protein.
6. gel viscosity is measured: with sample transfer in sample holder, constant temperature in 4 ℃ and the 37 ℃ of water-baths, adopt respectively 10 with 100rpm tachometric survey gelling before and after the viscosity of leptin-chitosan-mono methoxy polyethylene glycol hydrogel.
3) the external release experiment of leptin administration gel systems
To contain leptin (10
-5M) chitosan-mono methoxy polyethylene glycol administration slow release solution takes out 1ml immediately and places bag filter (molecular cut off 5KD) respectively after the gelling of 37 ℃ of constant temperature incubators; Put into vial; The PBS that in bottle, adds pH7.4, jolting in 37 ℃ of constant temperature oscillators (rotating speed 100rpm).At 5min, 15min, 45min, 120min, 300min, 600min, 1440min all takes out dissolution fluid respectively, adds 37 ℃ PBS, and the high effective liquid chromatography for measuring preparation is in the cumulative release amount of each time point.The same manner is with leptin (10
-5M) under PBS solution, carry out the cumulative release amount of high effective liquid chromatography for measuring at each time point.All under 20 ± 3 ℃ of conditions of indoor temperature, accomplish in all experiments.Mobile phase through 0.45 μ m microporous filter membrane negative pressure of vacuum filter, ultrasonic aerofluxus.The liquid of separating out is carried out HPLC successively detect behind 0.22 μ m membrane filtration, observe the external release of leptin.
The SPSS statistical software analyzes, and experimental data is represented with x ± s, every group of sample size n >=3.Group difference relatively adopts One-Way ANOVO to analyze the LSD method, and P＜0.05 thinks that difference has significance.
Result and discussion
1) expression and purification of leptin protein
Obtained purity and reached 90% leptin protein, satisfied experimental requirements, the electrophoretogram of its purification is seen Fig. 1.
2) it is good to have obtained temperature sensitivity, chitosan-mono methoxy polyethylene glycol gel delivery system that gel time is short, and its gel time is seen Fig. 2.
3) the external release experiment of leptin administration gel systems
To contain 10
-5The albumen that the chitosan of M leptin-mono methoxy polyethylene glycol administration slow release solution is dialysed and obtained after the gelling of 37 ℃ of constant temperature incubators; With the cumulative release amount of high effective liquid chromatography for measuring at each time point, with the same concentrations leptin in the PBS system, dialyse discharge relatively see Fig. 3.
Embodiment two leptin gel rubber sustained-release drug-supplying systems are used (joint cavity injection treatment bone loss)
Experiment material:
The female sd inbred rats of 24 10 SPF level raisings in age in week, superclean bench, syringe, scalpel, operating equipments such as mosquito forceps, 75% ethanol, 3% pentobarbital sodium, the above-mentioned sustained-release gel that contains leptin, leptin protein, chitosan-mono methoxy polyethylene glycol gel.
Experimental technique
With the female sd inbred rats that the SPF level in 24 10 ages in week is raised, be divided into 4 groups of called after A, B, C, D respectively immediately, 6 every group.All rats are all anaesthetized with 3% pentobarbital sodium (40mg/kg) intraperitoneal injection, except that D group row is opened merely the abdomen, and all the other 3 groups all capable bilateral oophorectomies, postoperative is put rat sub-cage rearing under room temperature, freely takes the photograph water.
After spay was raised and repaired for 3 weeks, experimental group A: at the chitosan-mono methoxy polyethylene glycol gel of rat intraarticular injection leptin, wherein the dosage of leptin was 3.5mg/kg, and injection once has altogether 3 times weekly.Experimental group B: only inject the leptin that Isodose is dissolved in normal saline, other condition is with experimental group A.Experimental group C: with the chitosan-mono methoxy polyethylene glycol gel of A group, do not contain leptin protein with dosage at the rat joint cavity injection.Experimental group D does not process, as contrast.
The preliminary experiment result shows that the bone density of experimental group A rat obviously is better than two groups of B, C.
The present invention obviously reduces the decomposition rate of pharmaceutical grade protein, through the half-life that the drug gel slow-released system improves pharmaceutical grade protein, improves the effectiveness of pharmaceutical grade protein.Chitosan of the present invention-mono methoxy polyethylene glycol gel rubber sustained-release system has temperature sensitive superperformance.
Claims (3)
1. leptin-chitosan-mono methoxy polyethylene glycol gel delivery system is characterized in that it is 10 that described drug-supplying system contains concentration
-5-10
-4The chitosan of the leptin of M, percentage by weight 3.0%-3.5%, 3.0%-3.5% mono methoxy polyethylene glycol and 0.01%-0.1% collagen protein.
2. leptin-chitosan according to claim 1-mono methoxy polyethylene glycol gel delivery system, its preparation method comprises following process:
1) preparation of mono methoxy polyethylene glycol solution: take by weighing mono methoxy polyethylene glycol, be dissolved in distilled water, sustained oscillation on the oscillating agitator makes its thorough dissolving, and processing concentration is 30% mono methoxy polyethylene glycol solution (w/v), membrane filtration, and low temperature storage is subsequent use.
2) preparation of chitosan solution: use acetic acid preparing chitosan solution, stirs after 2 days behind the chitosan solution autoclave sterilization with acquisition, use after being cooled to room temperature.
3) preparation of leptin-collagen solution: collagen is dissolved in the sodium hydroxide, and regulates pH value, add leptin again and make its dissolving to 7.0-7.4.
4) preparation of leptin-chitosan-mono methoxy polyethylene glycol gel: behind three kinds of solution ice bath 30min; Draw chitosan-acetic acid solution in container; After dripping leptin-collagen solution, stirring dropwise adds mono methoxy polyethylene glycol solution simultaneously fast, and continues to stir 20min.In the leptin-chitosan-mono methoxy polyethylene glycol gel systems that obtains, the concentration of leptin is 10
-5-10
-4M, chitosan mass concentration are 3.0%-3.5%, and mono methoxy polyethylene glycol concentration is 3.0%-3.5%, 0.01%-0.1% collagen protein.
3. leptin-chitosan according to claim 1-mono methoxy polyethylene glycol gel delivery system is characterized in that, described leptin gel rubber sustained-release drug-supplying system can be used as the local injection medicine, the treatment bone loss.
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Effective date of registration: 20180725 Address after: 100730 Wangfujing, Dongcheng District, Dongcheng District, Beijing Patentee after: Beijing Xiehe Hospital, Chinese Academy of Medical Sciences Address before: 100005 Peking Union Medical College Hospital, 1 Shuai Fu Garden, Dongcheng District, Beijing. Patentee before: Wu Zhihong |
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Effective date of registration: 20191015 Address after: 100176 Room 502, unit 1, building 6, No. 88, Kechuang 6th Street, Beijing Economic and Technological Development Zone Patentee after: Hezhe Technology Co., Ltd. Address before: 100730 Beijing city Dongcheng District Wangfujing Park No. 1 Patentee before: Beijing Xiehe Hospital, Chinese Academy of Medical Sciences |