CN102462662A - Liposome-encapsulated recombinant human ciliary neurotrophic factor - Google Patents
Liposome-encapsulated recombinant human ciliary neurotrophic factor Download PDFInfo
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- CN102462662A CN102462662A CN2010105528646A CN201010552864A CN102462662A CN 102462662 A CN102462662 A CN 102462662A CN 2010105528646 A CN2010105528646 A CN 2010105528646A CN 201010552864 A CN201010552864 A CN 201010552864A CN 102462662 A CN102462662 A CN 102462662A
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- cntf
- phospholipid
- monostalotetrahexosylgangliside
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Abstract
The invention provides a liposome-encapsulated recombinant human ciliary neurotrophic factor (rhCNTF) containing monosialo ganglioside (GM1) and a preparation method thereof. Particle diameters of liposome of the rhCNTF prepared by adopting the preparation method is evenly distributed within a range of 20 to 200nm, and the drug encapsulating rate is no less than 70 percent. An animal experiment proves that by adopting the rhCNTF liposome, the rhCNTF can pass through a blood brain barrier, and the rhCNTF liposome has the application value of curing diseases related to neuronal damages and degenerative diseases.
Description
Technical field
The present invention relates to a kind of method for preparing that contains recombination human ciliary neurotrophy factor (rhCNTF) liposome, liposome prescription and purposes.
Background technology
(Ciliary Neurotrophic Factor CNTF) extracts from ocular tissue's ciliary ganglion of Embryo Gallus domesticus at first CNTF, because of the survival that also can keep the chicken parasympathetic ganglion to the nutritious effect of ciliary nerves unit is gained the name.Find that after deliberation CNTF has multiple function, at nervous system, musculature, significant in the treatment of obesity and relevant disease thereof, wide clinical development prospect is arranged.But because the obstruct of blood brain barrier; It is limited that CNTF arrives the central nervous system; Hindered CNTF to be used for treatment of diseases such as Parkinson's disease, senile dementia; Can help drug molecule to pass through blood brain barrier through the liposome compound formulation, for CNTF medication in the central nervous system provides feasible approach.Because of the CNTF albumen toxic and side effects of native sequences is bigger, its clinical practice is affected.For seeking CNTF safely and effectively, people have worked out the mutant of multiple CNTF.Regeneron drugmaker has delivered the synthetic analog Axokine (AX15) of the CNTF that is used for treatment of obesity.AX15 is the triple mutant body of people CNTF: 17 Cys is replaced by Ala, and 63 Gln is replaced by Arg, and has lacked 15 aminoacid of C-terminal.CNTF compares with natural human, and AX15 has higher BA, better stability and solubility.A kind of effects of recombinant ciliary neurotrophic factor mutant (Chinese patent has been invented by Nat'l Pharmaceutical & Biological Products Control Institute; 200510097985.5; Open day: on April 19th, 2006); The 17th Cys of mutant sports Ser, and removes 16 aminoacid of C end, and this mutant is having better effects aspect treatment of obesity or disease relevant with obesity and the nerve injury relevant disease.A kind of effects of recombinant ciliary neurotrophic factor mutant (Chinese patent has been invented by Lanzhou Institute of Biological Products; 200710112387.X; Open day: on March 19th, 2008); 17 Cys of natural human CNTF gene are sported Ala, and 63 Gln sport Arg, aspect treatment common bariatric or the diabetes type obesity better effects are being arranged.
Known CNTF can get into blood brain barrier by trace, acts on the hypothalamus appestat, and the plain appearance of activity path plays the effect that reduces body weight.In addition, there are some researches show that direct injection CNTF can promote retinal ganglial cells (retinal ganglinecells, RGC) the RGC survival and the optic nerve regeneration of damage in the vitreous chamber.Weise (Neurobiol Dis; 2000,7:212-223) wait the means of utilizing gene therapy, with adenovirus (adenoviral vector; Ad) inject in the Mus vitreous body as the CNTF genophore; Can make CNTF mRNA and protein stabilized expression, continue 18 days at least, play the effect of pathological changes such as effective treatment neuratrophia.Yet people CNTF natural or reorganization can not effectively pass through blood brain barrier by blood circulation behind subcutaneous or intravenously administrable, realize the effect of treatment nervus centralis relevant diseases.The invention provides rhCNTF (A
17R
6315) situation in the preparation technology of liposome and the entering cerebral tissue thereof can realize that people CNTF effectively passes through blood brain barrier.The present invention is the basis with the cDNA of Axokine (AX15); On gene order, designed and had Nde I restriction enzyme site and ATG translation initiation codon; Downstream primer then has BamHI restriction enzyme site and TAA termination codon, all uses the escherichia coli preference codon, called after rhCNTF (A
17R
6315) (Recombinant Human CNTF, rhCNTF).Adopt expression vector plasmid pET-3c, recombinant expressed and obtain the rhCNTF of biologically active through efficiently purifying technology, it is successfully used to contain the liposome preparation research of CNTF.
Summary of the invention
The object of the present invention is to provide a kind of liposome that contains rhCNTF that can pass through blood brain barrier, thereby improve the neuronal damage disease relevant, like the therapeutic effect of Parkinson's disease, senile dementia, lateral sclerosis of spinal cord (ALS) etc. with degeneration.
For realizing above-mentioned purpose, the present invention is the basis with the cDNA of Axokine (AX15) and (the cDNA C end of wild type people CNTF is removed 15 aminoacid, increase its biological activity; Simultaneously 17 Cys are sported Ala; 63 Gln sport Arg), on gene order, designed and had restriction enzyme site and ATG translation initiation codon, downstream primer then has restriction enzyme site and TAA termination codon; All use escherichia coli preference codon, called after rhCNTF (A
17 R
6315).After artificial full gene is synthetic with Nde I and BamHI double digestion after, under the effect of T4 ligase, be connected transformed into escherichia coli DH5 α with the big fragment of pET-3c of Nde I and BamHI double digestion.With the recombinant plasmid transformed BL that contains the exogenous gene of AX15 after identifying
21(DE
3) plysS, the engineering bacteria of inducing screening to efficiently express.RhCNTF (A
17R
6315) purifying process comprises inclusion body processing, renaturation and recombinant protein purification process.
The invention provides a kind of method for preparing of the rhCNTF of containing liposome.The particle diameter of the liposome of the present invention's preparation is 20-200nm, and the envelop rate of rhCNTF is not less than 70%.Liposome is to be dispersed in the multilamellar microcapsule that forms in the water by phospholipid.Microcapsule is lipid bilayer for every layer, is separated by water between each layer.Liposome at first was used as pharmaceutical carrier in 1971 by Britain Lai Men people such as (Rymen).Liposome has biological degradability and biocompatibility as the carrier of medicine or other material, through delaying drug metabolism and removing, reducing the drug distribution volume, helps medicine and distributes to pathological tissues, and continuable drug release is provided.The liposome molecule of a 100nm can wrap up up to ten thousand drug molecules, and liposome can increase the transhipment amount of medicine at brain significantly.Liposome technology has been widely used in the medicine carrying of small-molecule drug and biopharmaceutical macromolecular drug as a kind of new formulation of medicine.
Liposome can be improved metabolism and the tissue distribution behavior of drug molecule in blood, reaches the purpose of slow release and targeting property.The prescription of liposome and method for preparing have had certain Maturity, and conventional prescription mainly contains the phospholipid and the cholesterol of different proportion, and method for preparing comprises thin-film ultrasonic method, reverse phase evaporation, vortex dispersion method etc.This patent is for realizing containing the brain target administration of CNTF liposome; With Monostalotetrahexosylgangliside (GM1) as the main component in the prescription of liposome; With certain phospholipid and cholesterol combination, prepare the unique liposome that contains CNTF again through thin-film ultrasonic method and squeezing and pressing method.Known phospholipid has various ways, like natural phospholipid and synthetic phospholipid.Natural phospholipid has the component inhomogeneity, and synthetic phospholipid is main with single component.The phospholipid that this patent adopts mainly refers to synthetic phospholipid, comprises DSPE, DSPC, DPPC, DMPG, DSPA, DPPG etc.
Description of drawings
The base of Fig. 1: rhCNTF (A17R6315) and aminoacid sequence contrast sketch map
Fig. 2: pET-rhCNTF engineering bacteria sequencing result.Wherein the cDNA of rhCNTF is a 50-617 position forward sequence, measures the result with implementation sequence and fits like a glove.
Fig. 3: rhCNTF expresses and the SDS-PAGE of purification analyzes collection of illustrative plates.(A) 1: express contrast; 2: the destination protein rhCNTF of abduction delivering.(B) rhCNTF behind the 1:Q column purification; 2: rhCNTF after the renaturation; 3: protein molecular weight standard.
The particle size determination analysis chart of Fig. 4 rhCNTF liposome.
The specific embodiment
The present invention will be described in more detail through specific embodiment for hereinafter, but protection scope of the present invention does not receive the restriction of embodiment.
Embodiment 1rhCNTF (A
17R
6315) acquisition of gene
Obtain CNTF cDNA sequence according to Gene Bank, the cDNA C end of wild type people CNTF is removed 15 aminoacid, increase its biological activity; Simultaneously 17 Cys are sported Ala, 63 Gln sport Arg, on gene order, have designed to have NdeI restriction enzyme site and ATG translation initiation codon; Downstream primer then has BamHI restriction enzyme site and TAA termination codon; All use the escherichia coli preference codon, full gene synthetic, and embed pUC18 carrier and called after pUC-CNTF.Corresponding base and amino acid sequence analysis such as Fig. 1
Structure and the expression of embodiment 2 plasmid pET-CNTF
Behind Nde I and BamHI double digestion; Be connected under the effect of T4 ligase with the big fragment of pET-3c of Nde I and BamHI double digestion; LB-Ampicillin is dull and stereotyped in transformed into escherichia coli DH5 α coating; Extract the positive colony plasmid, after Nde I and the evaluation correctly of BamHI double digestion, called after pET-CNTF.This recombinant expression plasmid transforms BL21 (DE3) plyss, obtains the engineering bacteria of recombinant expressed CNTF.IPTG abduction delivering screening high expressed bacterial strain is also preserved.Engineering bacteria sequencing result such as Fig. 2.Engineering bacteria abduction delivering such as Fig. 3 A.
The separation and purification of embodiment 3rhCNTF
Through experiment proof rhCNTF recombiant protein is that inclusion body is expressed.
1. the thalline of refrigerator being preserved was put into bacterial lysate with 1: 10, and is ultrasonic behind 37 ℃ of following stirring 4h, after 4 ℃ of coolings; By power P＜5 seconds 400W working times, intermittently 5 seconds ultrasonic 40 times, the centrifugal 10min of 12000r/min; Get the supernatant electrophoresis, inclusion body is further handled.
With inclusion body put into cleaning mixture A (20mmol/L Tris-HCl pH 8.0,5mmol/L EDTA, 2mol/L Urea, 1%TritonX-100) in, ultrasonic power P＜5 seconds 340W working times, intermittently 5 seconds ultrasonic 40 times.After stirring 30min, the centrifugal 15min of 12000r/min gets supernatant A 60 μ L and carries out SDS-PAGE.
3. inclusion body is put into cleaning mixture B (20mmol/L Tris-HCl pH 8.0; 4mol/L Urea, 1.0mol/L NaCl) in, ultrasonic power P＜5 seconds 340W working times; 5 seconds ultrasonic 40 times (carbamide and bacterium liquid are fully acted on) intermittently; After stirring 30min, the centrifugal 15min of 12000r/min gets supernatant B 60 and carries out SDS-PAGE.
4. dissolving inclusion body: add solubilization of inclusion bodies liquid (25mmol/L Tris-HCl pH 8.0,8mol/L Urea, 2mmol/L EDTA with 10% of original bacteria liquid volume; 1/2000B-ME), 37 ℃ of incubation 3h, 4 ℃ of 8～10h fully dissolve inclusion body; Centrifugal 12000r/min 15min abandons insoluble matter.
5. dilution refolding: progressively add the renaturation solution (25mmol/L Tris-HCl pH 8.0) of 10 times of volumes in the solubilization of inclusion bodies liquid, place 4 ℃, 100r/min to stir, carry out dilution refolding.
6.Q-Sepharose column chromatography purification: the buffer balance of 25mmol/L Tris-HCl pH 8.0, contain the buffer solution for gradient elution of 0.5mol/L NaCl, collect the rhCNTF eluent.
7. rhCNTF reaches 98.0% (Fig. 3 B) through SDS-PAGE and HPLC purity assay behind the purification.ED50 alive is 1.2ng/mL through TF-1 cell in vitro biometric.
The preparation of embodiment 4rhCNTF liposome
Get GM1250mg, PHOSPHATIDYL ETHANOLAMINE (DSPE) 500mg and cholesterol 1000mg, be dissolved in the 50mL organic solvent.Abundant dissolving back rotary evaporation, treat that residual organic solvent is volatilized fully after, rhCNTF (10mmol/LpH 7.4 phosphate solutions) the 100mL lipin dissolving body film with 2.5mg/mL makes aqueous suspension.Pop one's head in ultrasonic (power 20-50W, ultrasonic time 5s, blanking time 4s, ultrasonic number of times 10-40 time), cross 0.22 μ m filter membrane behind the high pressure homogenizer homogenizing, promptly get the rhCNTF liposome.Adopt the laser particle analyzer to measure rhCNTF liposome particle diameter.Particle size distribution range is 20-200nm, and mean diameter is 85.0nm (Fig. 4).
The mensuration of embodiment 5 envelop rates
Get 1mL rhCNTF liposome, add 1: behind the 10DDW, the centrifugal 2h of 100000r/min (CP100MX of Hitachi superspeed refrigerated centrifuge).Get cleer and peaceful liposome deposition, the liposome deposition is with 10mL breakdown of emulsion liquid (1%SDS, 10%TritonX-100) thorough breakdown of emulsion.HPLC base peak area according to rhCNTF; Calculate the total content of rhCNTF in deposition and the supernatant, the rhCNTF liposome encapsulation for preparing with liposome encapsulation formula computational envelope rate
is 82.5%.
Embodiment 6rhCNTF standard curve
Get rhCNTF stock solution 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 250ng/mL, 500ng/mL, 750ng/mL, 1000ng/mL and add ELISA Plate and measure, get Human CNTF ELISA (R&D company) test kit sample diluting liquid simultaneously as blank.450nm draws formula according to OD value and sample concentration after measuring the OD value: Y=66.011X-1.6658 (Y is a concentration, and X is the OD value), and R=0.999, the range of linearity is 10-1000ng/mL.
The experiment of embodiment 7 precision
HPLC measures the experiment of rhCNTF concentration precision: it is an amount of that precision is measured the rhCNTF storing solution, and using mobile phase A to be diluted to mass concentration is 100,250,500 μ g/mL solution.Repeat sample introduction 5 times, sample size in 1d with in 1 week: 20 μ L, calculate and in a few days see table 1 with day to day precision.
ELISA measures the experiment of rhCNTF content precision: it is an amount of that precision is measured the rhCNTF storing solution, is diluted to 100,250,500ng/mL solution with Human CNTF ELISA test kit sample diluting liquid.In 1d and replication 5 times in 1 week, calculating is in a few days seen table 2 with day to day precision.
Table 1HPLC measure rhCNTF concentration in a few days and day to day precision
Table 2ELISA measure rhCNTF content in a few days and day to day precision
Embodiment 8rhCNTF extraction recovery
Dry ice is put to death 18 Balb/C mices; Getting brain cleans 3-4 time with PBS; Select blank brain, blank brain+100ng/mL rhCNTF, blank brain+150ng/mL rhCNTF, blank brain+200ng/mL rhCNTF, blank brain+250ng/mL rhCNTF for use, after blank brain+each 1mL of 300ng/mL rhCNTF carries out homogenate; Add 1mLHuman CNTF ELISA test kit sample diluting liquid; The centrifugal 10min of 10000r/min gets supernatant with HumanCNTF ELISA kit measurement OD value, according to the standard curve Equation for Calculating response rate.The rhCNTF extraction recovery is seen table 3.
Table 3rhCNTF extraction recovery
Embodiment 9rhCNTF liposome gets into cerebral tissue and measures
Get 78 Balb/C mices; Be divided into 4 groups; Press 5mg/kg rhCNTF tail vein injection A group: rhCNTF aqueous solution (10mmol/LpB pH7.4), B group: high dose 5mg/kg rhCNTF liposome, C group: low dosage 1.5mg/kg rhCNTF liposome; The D group: normal saline, put to death mice at 2h, 6h, 12h, 24h dry ice.Getting brain cleans 3-4 time with PBS; Fully remove blood and brain vascular surface; With 0.5mL Human CNTF ELISA test kit sample diluting liquid homogenate liquid is washed out after the Potter-Elvehjem Tissue Grinders homogenate; Centrifugal 10000r/min, 10min get supernatant and measure with the ELISA test kit, calculate the content of CNTF in every Borneo camphor tissue with the ELISA standard curve.
RhCNTF content in mouse brain compares behind table 4rhCNTF aqueous solution group and the rhCNTF liposome group injection mice
*P＜0.01vs. aqueous solution group.
Can draw from above result: have trace to get into the cerebral tissue without the rhCNTF of liposome, and rhCNTF is after liposome, the rhCNTF that gets in the cerebral tissue significantly improves more than 5 times.
Claims (8)
1. method for preparing and a prescription and purposes of recombination human ciliary neurotrophy factor (rhCNTF) being carried out liposome.It is characterized in that the rhCNTF of liposome form can more effectively pass through blood brain barrier.
2. the liposome prescription in the claim 1 is characterised in that and contains Monostalotetrahexosylgangliside (GM1), phospholipid and cholesterol, and its ratio is 1: 4: 8 to 1: 2: 4.
3. the content of Monostalotetrahexosylgangliside in liposome is 0.25%～1.0% in the claim 2.
4. phospholipid is any among synthetic phospholipid DSPE, DSPC, DPPC, DMPG, DSPA, the DPPG in the claim 2.
5. the rhCNTF in the claim 1 refers to have CNTF analog or the recombiant protein of mutant of natural CNTF biological activity more than 70%.
6. 17 of the preferred CNTF of rhCNTF Cys sport Ala in the claim 3, and 63 Gln sport Arg, and have lacked 15 amino acid whose rh-CNTF (A of C-terminal
17R
6315).
7. the method for preparing of liposome CNTF may further comprise the steps in the claim 1:
A) getting a certain amount of Monostalotetrahexosylgangliside (GM1), synthetic phospholipid and cholesterol is dissolved in the methanol chloroform by corresponding proportion (70: 30, in solvent V/V), rotary evaporation was processed the single or multiple lift lipid film.
B) contain 0.02-1.0%rhCNTF phosphate solution (pH5.5-8.0) and add in the lipid film, the liposome of supersound process preparation parcel rhCNTF.
C) the rhCNTF liposome is extruded device through high pressure, the liposome suspension of preparation 20-200nm particle diameter.
D) through 0.22 μ m membrane filtration, process aseptic liposome solutions.
E) add corresponding adjuvant and process preparations such as injection, lyophilized powder, microsphere, microcapsule.
8. claim 1 and the 7 described rhCNTF liposomees that contain can be used for being prepared into the medicine of the relevant aspects such as disease of neuronal damage or nerve retrograde affection.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106309410A (en) * | 2016-10-14 | 2017-01-11 | 珠海赛隆药业股份有限公司 | Monosialotetrahexosylganglioside sodium slow-release capsule and preparation method thereof |
| CN108272822A (en) * | 2018-01-18 | 2018-07-13 | 南京师范大学 | It is a kind of breast polar lipid extracting method and its application |
| CN113304276A (en) * | 2021-06-04 | 2021-08-27 | 沈阳药科大学 | Liposome modified by monosialotetrahexosylganglioside, preparation method and freeze-drying application thereof |
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| CN1195286A (en) * | 1995-07-14 | 1998-10-07 | 迪普技术公司 | Epidural administration therapeutic compounds with sustained rate of release |
| WO2004015113A2 (en) * | 2002-08-07 | 2004-02-19 | Delta Biotechnology Ltd. | Albumin-fused ciliary neurotrophic factor |
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2010
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1195286A (en) * | 1995-07-14 | 1998-10-07 | 迪普技术公司 | Epidural administration therapeutic compounds with sustained rate of release |
| WO2004015113A2 (en) * | 2002-08-07 | 2004-02-19 | Delta Biotechnology Ltd. | Albumin-fused ciliary neurotrophic factor |
Non-Patent Citations (2)
| Title |
|---|
| 刘军等: "自制脂质体包裹神经生长因子通过血脑屏障的初步研究", 《南京医科大学学报（自然科学版）》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106309410A (en) * | 2016-10-14 | 2017-01-11 | 珠海赛隆药业股份有限公司 | Monosialotetrahexosylganglioside sodium slow-release capsule and preparation method thereof |
| CN106309410B (en) * | 2016-10-14 | 2019-01-08 | 珠海赛隆药业股份有限公司 | A kind of monosialotetrahexose ganglioside sodium spansule and preparation method thereof |
| CN108272822A (en) * | 2018-01-18 | 2018-07-13 | 南京师范大学 | It is a kind of breast polar lipid extracting method and its application |
| CN108272822B (en) * | 2018-01-18 | 2021-04-13 | 南京师范大学 | Extraction method and application of milk polar lipid |
| CN113304276A (en) * | 2021-06-04 | 2021-08-27 | 沈阳药科大学 | Liposome modified by monosialotetrahexosylganglioside, preparation method and freeze-drying application thereof |
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Application publication date: 20120523 |