CN102516391A - Neuropilin-1 ligand polypeptide-polyethylene glycol-phospholipid composite, its active targeting liposome vector system and preparation method thereof - Google Patents
Neuropilin-1 ligand polypeptide-polyethylene glycol-phospholipid composite, its active targeting liposome vector system and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to a neuropilin-1 ligand polypeptide-polyethylene glycol-phospholipid composite, its active targeting liposome vector system and a preparation method thereof. The liposome vector system comprises the following components: a: phospholipid, b: cholesterol, c: methoxy polyethylene glycol-phospholipid composite (with the molecular weight of methoxy polyethylene glycol ranging from 400 to 6000), and d: a RPAKPAR sequence-containing polypeptide-polyethylene glycol-phospholipid composite (with the molecular weight of polyethylene glycol ranging from 400 to 8000). And for the components, a and b are in a molar ratio of 5:1-1:5, a and c are in a molar ratio of 1000:1-1000:100, and a and d are in a ratio of 1000:0.1-1000:100. The system can make a liposome passively drained into a lymphatic system through subcutaneous interstitial injection or intramuscular injection, and can make the liposome targeted to a lymphatic metastasis lesion through the mediation effect of RPAKPAR. The system can also make the liposome targeted to a primary tumor and a hematogenous metastasis lesion directly through intravenous injection administration, through a tumor EPR (electron paramagnetic resonance) effect and the mediation effect of RPAKPAR. The liposome vector system of the invention can be used for targeted drug delivery in diagnosis or treatment of prostate cancer primary tumors and metastatic tumors.
Description
Technical field
The present invention relates to a kind of neuropil albumen-1 ligand polypeptide-polyoxyethylene glycol-phospholipid complex, its active target liposomes carrier system and preparation method thereof.
Background technology
Prostate cancer is the modal malignant tumour of male reproductive system, and morbidity increased with the age, and its sickness rate has tangible areal variation, and the European and American areas is higher.It is reported to be only second to lung cancer, is second of cancer mortality the male sex.Along with the increase of aging population degree, the Chinese male sickness rate is obvious increase trend in recent years: Chinese prostate cancer sickness rate in 1993 rose to 4.55 people for 1.71 patients of annual per 100,000 philtrums to 2000.Because China is to the understanding of prostate cancer and pay attention to inadequately, and the getting up early prostate cancer has no symptom, is difficult for causing people's attention, so the prostate cancer that China finds is late case mostly, lymphatic metastasis or blood road has taken place shifted, and forms MET.To the prostate cancer patients with terminal; Operative treatment has been difficult to obtain better result of treatment, thus generally take external radiotherapy of extend range or chemotherapy means, but these two kinds of treatment means whole body toxic side effect are big; Conventional chemotherapy does not have the target effect to lesions position, and result of treatment is not good.Therefore, prostate cancer is carried out targeted therapy and have the important clinical realistic meaning.
In all nanometer delivery systems; Because the liposome membrane material is formed identical with cytolemma; Its biological safety problem is less, and is prone to advantages such as preparation, easy modification, particle diameter easy to control, high, the easy suitability for industrialized production of drug loading owing to it, and liposome is the maximum nano medicament carrying system of clinical application.Have launch such as Evacet, daunorubicin liposome and AM Bison at present, be mainly used in the targeted therapy of tumour or fungi infestation.The existing liposome overwhelming majority is based on passive target and plays a role, and target efficient is lower.Tumour active target liposomes delivery system based on the polypeptide mediation becomes nanometer delivery system and oncotherapy hot research fields gradually because of tumour being shown superior target property and growth-inhibiting effect.But at present tumour initiatively targeting drug delivery system from its intravenously administrable to process that tumour cell engages, still receive the restriction of two big barriers---tumor vessel barrier and tumor epithelial cell barrier, this problem has received investigator's common concern.There is the investigator to filter out some and has the targeted molecular that penetrates tumor vessel and tumor epithelial cell ability through the polypeptide triage techniques; Use it for mediation drug molecule or nanometer delivery system and get into tumor tissues; Obtain good effect, but do not seen the report that is used for liposome delivery system targeting drug delivery.Bibliographical information is not seen in the biological safety evaluation of the liposome of this type of polypeptide and modification thereof yet, and this also is the initiatively common problems of target liposomes delivery system clinical application of all tumours.
Summary of the invention
One of the object of the invention is to provide a kind of neuropil albumen-1 ligand polypeptide-polyoxyethylene glycol-phospholipid complex.
Two of the object of the invention is to provide a kind of active target liposomes carrier system that contains mixture.
Three of the object of the invention is to provide the preparation method of this liposome vectors system.
For achieving the above object, the present invention adopts following technical scheme:
A kind of neuropil albumen-1 ligand polypeptide-polyoxyethylene glycol-phospholipid complex; It is characterized in that this mixture by neuropil albumen-1 ligand polypeptide, polyoxyethylene glycol and phosphatide three parts through the covalently bound linear block copolymers that forms; Wherein the mol ratio of neuropil albumen-1 ligand polypeptide, polyoxyethylene glycol and phosphatide is 1:1:1, and the aminoacid sequence of described neuropil albumen-1 ligand polypeptide is: RPAKPAR.
The weight-average molecular weight of above-mentioned polyoxyethylene glycol can be 400 ~ 8000.
Above-mentioned polyoxyethylene glycol weight-average molecular weight can be 2000 ~ 5000.
Above-mentioned phosphatide can be DSPE, two palmityl phosphatidylethanolamines, DOPE, hydrogenated soya phosphatide acyl thanomin, hydrogenation egg phosphatide acyl thanomin, soybean phospholipid acyl thanomin or egg phosphatide acyl thanomin.
A kind of active target liposomes carrier system contains above-mentioned neuropil albumen-1 ligand polypeptide-polyoxyethylene glycol-phospholipid complex, it is characterized in that consisting of of this carrier system:
A. phosphatide;
B. SUV;
C. methoxy poly (ethylene glycol)-phospholipid complex;
D. neuropil albumen-1 ligand polypeptide-polyoxyethylene glycol-phospholipid complex;
Mol ratio between above-mentioned each composition is: a:b=5:1 ~ 1:5, a:c=1000:1 ~ 1000:100, a:d=1000:0.1 ~ 1000:100.
The described phosphatide of above-mentioned component a is: egg phosphatide; The hydrogenated soya phosphatide phatidylcholine; The hydrogenation Yolk lecithin; Two bay phosphatidyl cholines; Dimyristoyl phosphatidyl choline; DPPC; DSPC; 1-mnyristoyl-2-palmitoylphosphatidyl choline; 1-palmityl-2-stearyl phosphatidylcholine; 1-stearyl-2-palmitoylphosphatidyl choline; 1-palmityl-2-oleoyl phosphatidylcholine; The inferior oleoyl phosphatidylcholine of 1-stearyl-2-; The dioleoyl phospholipid phatidylcholine; The hydrogenation DPPC; DSPC; Two mnyristoyl phosphatidic acids; Two mnyristoyl phosphatidic acids; Two palmityl phosphatidic acids; Two palmityl phosphatidic acids; G 12S3P; Two mnyristoyl phosphatidylethanolamines; Two palmityl phosphatidylethanolamines; Kephalin acyl Serine; Two mnyristoyl phosphatidylserines; Two palmityl phosphatidylserines; Egg phosphatide acyl glycerine; Two lauryl POPGs; GLYCEROL,DIMYRISTOYL PHOSPHATIDYL; Two palmityl POPGs; The distearyl POPG; DOPG; The brain sphingophospholipid; Two palmityl sphingophospholipid or distearyl sphingophospholipid.
Phosphatide in above-mentioned methoxy poly (ethylene glycol)-phospholipid complex can be DSPE, two palmityl phosphatidylethanolamines, DOPE, hydrogenated soya phosphatide acyl thanomin, hydrogenation egg phosphatide acyl thanomin, soybean phospholipid acyl thanomin or egg phosphatide acyl thanomin.
Methoxy poly (ethylene glycol) weight-average molecular weight in above-mentioned methoxy poly (ethylene glycol)-phospholipid complex can be 400 ~ 6000.
A kind of method for preparing above-mentioned active target liposomes carrier system is characterized in that the concrete steps of this method are: take by weighing above-mentioned each component respectively and be dissolved in and prepare even lipid film, vacuum-drying in the chloroform; The ammoniumsulphate soln that adds 0.155M again, concussion obtains liposome turbid liquor in 20-70 ℃ of water-bath; In 20-70 ℃ of water-bath, pushed 400,200,100 and the 50nm nucleopore membranes more successively, blank liposome, be the active target liposomes carrier system that contains neuropil albumen-1 ligand polypeptide-polyoxyethylene glycol-phospholipid complex.
The preparing method's of above-mentioned neuropil albumen-1 ligand polypeptide-polyoxyethylene glycol-phospholipid complex concrete steps are:
A. be that tertbutyloxycarbonyl-l-arginine (p-toluenesulfonyl)-acetparaminosalol benzyl ester Boc-Arg (Tos)-PAM resin of 0.6mmol/g is used N with substitution value, dinethylformamide DMF swelling is drained; Slough Boc protection base with trifluoroacetic acid TFA again, take out TFA;
B. use benzotriazole-N, N, N', DMF solution and the N of N'-tetramethyl-urea hexafluorophosphate HBTU, N-diisopropylethylamine DIEA activation tertbutyloxycarbonyl-L-Ala Boc-Ala gets the Boc-Ala activation solution;
C. with the Boc-Ala activation solution of step a gained resin with DMF washing back adding step b gained, 25 ℃ of joltings were reacted 25 minutes, took out dereaction liquid, and used the DMF washing resin;
D. repeating step a-c connects all the other amino acid in order by the CRPAKPAR sequence; Reaction finishes after scouring resin, TFA deprotection base, vacuum-drying;
E. the resin of step e gained is put into polypeptide cutting pipe, add an amount of P-cresol, feed HF then, ice bath stirring reaction 1 hour; Reaction finishes the back decompression and takes out HF in the pipe, and raffinate is with an amount of ice ether sedimentation, crosses to filter deposition and with icing the ether washing precipitation; Deposition is crossed and is filtered filtrating again with the TFA dissolving; Filtrating is precipitated in the ice ether again, filters, and filter residue redissolves with water, and freeze-drying gets the pure article of CRPAKPAR;
F. the CRPAKPAR that step e is obtained is dissolved in the PBS solution of pH7.0; Get maleimide-polyoxyethylene glycol-phospholipid complex Mal-PEG-DSPE and be dissolved in DMF; Stirring reaction reacts completely to Mal-PEG-DSPE; Remove excessive CRPAKPAR and DMF, lyophilize obtains the neuropil albumen-1 ligand polypeptide-polyoxyethylene glycol-phospholipid complex of straight chain.
RPAKPAR is a kind of straight chain seven peptides that obtain through the display technique of bacteriophage screening, is the part of neuropil albumen-1.Research shows that neuropil albumen-1 has high expression level at prostate cancer tumour cell and tumor vascular endothelial cell surface, and does not have expression or hang down expression in healthy tissues.RPAKPAR shows pathoklisis to the tumour cell and the tumor vessel of neuropil albumen-1 high expression level, and has the ability that the mediation phage penetrates tumor vessel entering tumor tissues inside.Do not see at present the report that utilizes RPAKPAR polypeptide mediation nanometer delivery system to be used for prostate cancer and MET targeted therapy thereof as yet.
It is synthetic through solid-phase synthesis to contain the RPAKPAR polypeptide of sequence, and HPLC (HPLC) and MS (mass spectroscopy) characterize its structure.
Polypeptide-polyoxyethylene glycol-the phospholipid complex that contains the RPAKPAR sequences polypeptide; Synthetic in the following manner: containing the RPAKPAR polypeptide of sequence provides a free sulfhydryl groups; Polyoxyethylene glycol-phosphatide provides a dimaleoyl imino; Specific reaction takes place and connects into covalent complex in both, characterizes its structure through nucleus magnetic resonance (NMR) after synthesizing.
Liposome employing rotary evaporation-film aquation-pushed embrane method to prepare.A certain proportion of a, b, c, d are dissolved in chloroform, adopt rotary evaporation-film aquation legal system to be equipped with the liposome (RPAKPAR-liposome) that RPAKPAR modifies, reduce the liposome particle diameter, obtain liposome with the method for pushing film.The laser scattering method size distribution.Its median size is 30 ~ 1000nm.
The present invention is through the fluorescent tracing liposome, and the examination tumour cell is respectively to the RPAKPAR-liposome of year Fluoresceincarboxylic acid (FAM) and the picked-up of conventional liposome, and the result confirms that RPAKPAR gets into tumour cell at the external liposome that can mediate.
Through being model drug with the Zorubicin; Investigated RPAKPAR-liposome/DOX in the growth in vitro restraining effect of PC-3 prostate cancer cell and the body to the tumor growth restraining effect of prostate cancer, show that the modification of RPAKPAR can significantly increase the growth-inhibiting effect of Evacet to prostate cancer cell and tumor tissues.
Result of study prompting, the present invention's preparation contain active target liposomes carrier system that the RPAKPAR polypeptide of sequence modifies can be used for target prostate cancer and MET thereof.This system can through subcutis gap injection or intramuscular injection with the passive drainage of liposome to lymphsystem, through mediation target to the lymphatic metastasis focus of RPAKPAR.This system also can directly pass through intravenous administration, through tumour EPR effect and RPAKPAR mediation with its target to primary tumo(u)r and blood road metastatic lesion.This liposome vectors system can be used as the drug targeting of prostate cancer primary tumo(u)r and metastatic tumour diagnosis or treatment and sends.
Description of drawings
Fig. 1 is the HPLC collection of illustrative plates of CRPAKPAR
Chromatographic process: chromatographic column: Diamonsil C18 5 μ m 200 * 4.6mm enlightening horse; Mobile phase A: 0.1%TFA H
2O; Mobile phase B: 0.1%TFA acetonitrile; Elution program: 0-2min 5%B, 2 ~ 32min 5%-65%B, 32 ~ 33min, 65% ~ 90%B, 33 ~ 36min 90%B; Wavelength: UV214nm; Flow velocity: 0.7ml/min; Column temperature: 40 ℃;
Fig. 2 is the mass spectrum of CRPAKPAR;
Fig. 3 is RPAKPAR-PEG-DSPE's
1
The H-NMR collection of illustrative plates;
Fig. 4 is RPAKPAR-liposome/FAM and liposome/FAM size distribution figure;
Fig. 5 is that RPAKPAR-liposome/FAM and liposome/FAM are absorbed photo by the PC-3 tumour cell;
Fig. 6 is that RPAKPAR-liposome/DOX and liposome/DOX suppress effect to PC-3 prostate cancer tumor cell extracorporeal growth.
Embodiment
To help further to understand the present invention through following embodiment, but not limit content of the present invention.
Synthetic, the purifying of embodiment 1:CRPAKPAR and sign
Take by weighing Boc-Arg (Z)-PAM resin 0.4167g (substitution value 0.6mmol/g) in connecing the peptide bottle, resin, is drained after 20 minutes with DMF (N, dinethylformamide) swelling.Add TFA (trifluoroacetic acid) stirring reaction of about twice resin volume, take out TFA, add TFA more once, slough Boc protection base with the method operation.With DMF solution and DIEA (N, N-diisopropylethylamine) the activated b oc-Ala of HBTU (benzotriazole-N, N, N', N'-tetramethyl-urea hexafluorophosphate), resin adds the Boc-Ala activation solution with DMF washing back, the jolting reaction.Reaction is taken out dereaction liquid after finishing, and uses the DMF washing resin.Connect all the other amino acid with aforesaid method in order by the CRPAKPAR sequence subsequently.Reaction finishes the back and presses preceding method washing resin, TFA deprotection base.Use DMF, DCM/MeOH (methylene chloride, 1/1, v/v) washing resin, vacuum-drying successively.
Resin is put into polypeptide cutting pipe, add an amount of P-cresol, feed HF then, ice bath stirring reaction 1 hour.Reaction finishes the back decompression and takes out HF in the pipe, and raffinate is with an amount of ice ether sedimentation, crosses to filter deposition and with icing ether washing precipitation 3 times.Deposition is crossed and is filtered filtrating again with the TFA dissolving.Filtrating is precipitated in the ice ether again, and sand core funnel filters, and abandons filtrating; With water redissolution deposition, freeze-drying gets the pure article of CRPAKPAR, with HPLC and MS it is characterized; Referring to Fig. 1 and Fig. 2, calculating its molecular weight by the mass spectrum result is 898.10 to be consistent with theoretical molecular.The result shows that CRPAKPAR synthesizes successfully.
Embodiment 2:RPAKPAR-PEG
3350
Synthetic, the purifying of-DSPE and sign
The CRPAKPAR that above-mentioned steps is obtained is dissolved in (pH7.0) in the PBS solution, gets Mal-PEG
3350-DSPE (maleimide-polyoxyethylene glycol (molecular weight 3350)-phospholipid complex) is dissolved in DMF, and both mix back magnetic agitation reaction, and TLC (thin layer chromatography) monitoring reaction is treated Mal-PEG
3350Stopped reaction after-DSPE reacts completely, excessive CRPAKPAR and DMF remove through dialysis (molecular weight cut-off 3.5kDa).Lyophilize obtains the RPAKPAR-PEG of straight chain
3350-DSPE, NMR characterizes its structure, and referring to Fig. 3, A is Mal-PEG among the figure
3350The nuclear magnetic spectrum of-DSPE, B are RPAKPAR-PEG
3350The nuclear magnetic spectrum of-DSPE can be found out by figure, and A figure demonstrates the maleimide peak, and this peak disappears among the B figure, and all the other peaks remain unchanged basically, show Mal-PEG
3350Maleimide base group among the-DSPE reacts with RPAKPAR.The result shows, Mal-PEG
3350-DSPE collection of illustrative plates shows the characteristic peak of Mal at the 6.67ppm place, and RPAKPAR-PEG
3350This peak disappears on-DSPE the collection of illustrative plates, and RPAKPAR-PEG is described
3350-DSPE synthesizes successfully.
Embodiment 3:RPAKPAR-PEG
3350
Synthetic, the purifying of-DPPE and sign
The CRPAKPAR that above-mentioned steps is obtained is dissolved in (pH7.0) in the PBS solution, gets Mal-PEG
3350-DPPE (maleimide-polyoxyethylene glycol (molecular weight 3350)-phospholipid complex) is dissolved in DMF, and both mix back magnetic agitation reaction, and TLC (thin layer chromatography) monitoring reaction is treated Mal-PEG
3350Stopped reaction after-DPPE reacts completely, excessive CRPAKPAR and DMF remove through dialysis (molecular weight cut-off 3.5kDa).Lyophilize obtains the RPAKPAR-PEG of straight chain
3350-DPPE, NMR characterizes its structure, and referring to Fig. 3, A is Mal-PEG among the figure
3350The nuclear magnetic spectrum of-DPPE, B are RPAKPAR-PEG
3350The nuclear magnetic spectrum of-DPPE can be found out by figure, and A figure demonstrates the maleimide peak, and this peak disappears among the B figure, and all the other peaks remain unchanged basically, show Mal-PEG
3350Maleimide base group among the-DPPE reacts with RPAKPAR.The result shows, Mal-PEG
3350-DPPE collection of illustrative plates shows the characteristic peak of Mal at the 6.67ppm place, and RPAKPAR-PEG
3350This peak disappears on-DPPE the collection of illustrative plates, and RPAKPAR-PEG is described
3350-DPPE synthesizes successfully.
Embodiment 4:RPAKPAR-PEG
4500
Synthetic, the purifying of-DSPE and sign
The CRPAKPAR that above-mentioned steps is obtained is dissolved in (pH7.0) in the PBS solution, gets Mal-PEG
4500-DSPE (maleimide-polyoxyethylene glycol (molecular weight 4500)-phospholipid complex) is dissolved in DMF, and both mix back magnetic agitation reaction, and TLC (thin layer chromatography) monitoring reaction is treated Mal-PEG
4500Stopped reaction after-DSPE reacts completely, excessive CRPAKPAR and DMF remove through dialysis (molecular weight cut-off 3.5kDa).Lyophilize obtains the RPAKPAR-PEG of straight chain
4500-DSPE, NMR characterizes its structure, and referring to Fig. 3, A is Mal-PEG among the figure
4500The nuclear magnetic spectrum of-DSPE, B are RPAKPAR-PEG
4500The nuclear magnetic spectrum of-DSPE can be found out by figure, and A figure demonstrates the maleimide peak, and this peak disappears among the B figure, and all the other peaks remain unchanged basically, show Mal-PEG
4500Maleimide base group among the-DSPE reacts with RPAKPAR.The result shows, Mal-PEG
4500-DSPE collection of illustrative plates shows the characteristic peak of Mal at the 6.67ppm place, and RPAKPAR-PEG
4500This peak disappears on-DSPE the collection of illustrative plates, and RPAKPAR-PEG is described
4500-DSPE synthesizes successfully.
Preparation and the sign of embodiment 5:RPAKPAR-liposome/FAM
Liposome membrane material prescription consists of HSPC (hydrogenated soya phosphatide)/Chol (SUV)/mPEG
2000(55:45:2, mol/mol), the PEG liposome membrane material prescription that RPAKPAR modifies is HSPC/Chol/mPEG to-DSPE (methoxy poly (ethylene glycol) (molecular weight 2000)-DSPE mixture)
2000-DSPE/ RPAKPAR-PEG
3350-DSPE (55:45:2:0.5, mol/mol).Take by weighing above-mentioned mould material and be dissolved in chloroform, the decompression rotary evaporation is removed organic solvent, gets even lipid film, vacuum-drying 24 hours.Add FAM aqueous solution aquation, 60 ℃ of water-baths were shaken 2 hours, got liposome turbid liquor.In 60 ℃ of water-baths, use high pressure homogenizer (if the liposome volume is less than 10 mL and then uses the miniature device of extruding instead) successively liposome to be pushed 400,200,100 and the 50nm nucleopore membranes, its particle diameter is reduced.Be that elutriant is crossed sephadex G-50 chromatography column and separated and to remove non-encapsulated FAM then with saline water, liposome.Liposome is used the dynamic light scattering determination particle diameter, shows that particle diameter all is about 90nm.
Preparation and the sign of embodiment 6:RPAKPAR-liposome/FAM
Liposome membrane material prescription consists of DPPC (DPPC)/Chol (SUV)/mPEG
3000(55:45:2, mol/mol), the PEG liposome membrane material prescription that RPAKPAR modifies is DPPC/Chol/mPEG to-DSPE (methoxy poly (ethylene glycol) (molecular weight 3000)-DSPE mixture)
2000-DSPE/ RPAKPAR-PEG
4500-DSPE (55:45:2:0.5, mol/mol).Take by weighing above-mentioned mould material and be dissolved in chloroform, the decompression rotary evaporation is removed organic solvent, gets even lipid film, vacuum-drying 24 hours.Add FAM aqueous solution aquation, 60 ℃ of water-baths were shaken 2 hours, got liposome turbid liquor.In 60 ℃ of water-baths, use high pressure homogenizer (if the liposome volume is less than 10 mL and then uses the miniature device of extruding instead) successively liposome to be pushed 400,200,100 and the 50nm nucleopore membranes, its particle diameter is reduced.Be that elutriant is crossed sephadex G-50 chromatography column and separated and to remove non-encapsulated FAM then with saline water, liposome.Liposome is used the dynamic light scattering determination particle diameter, shows that particle diameter all is about 80nm.
Preparation and the sign of embodiment 7:RPAKPAR-liposome/DOX
Conventional liposome mould material prescription consists of HSPC/Chol/mPEG
2000(55:45:2, mol/mol), the liposome membrane material prescription that RPAKPAR modifies is HSPC/Chol/mPEG to-DSPE
2000-DSPE/ RPAKPAR-PEG
3350-DSPE (55:45:2:1, mol/mol).Take by weighing above-mentioned mould material respectively and be dissolved in chloroform, the decompression rotary evaporation is removed chloroform, gets even lipid film, vacuum-drying 24h.The 0.155M ammoniumsulphate soln that adds certain volume, 60 ℃ of water-bath concussion 2h get liposome turbid liquor.In 60 ℃ of water-baths, use high pressure homogenizer (if the liposome volume is less than 10mL and then uses the miniature device of extruding instead) successively liposome to be pushed 400,200,100 and the 50nm nucleopore membranes, blank liposome.With saline water is eluent, and the blank liposome wash-out is changed outer water through Sephadex G-50 gel column.Add Zorubicin physiological salt soln, 60 ℃ of water-bath 20min according to medicine fat than 1:10 (w/w).Through Sephadex G-50 gel column, remove free drug with the saline water wash-out, get Evacet.Use the dynamic light scattering determination particle diameter, referring to Fig. 4, liposome/FAM and RPAKPAR-liposome/FAM size distribution figure can be known by figure, both size and the no significant difference that distributes.Show that particle diameter all is about 90nm.
Preparation and the sign of embodiment 8:RPAKPAR-liposome/DOX
Conventional liposome mould material prescription consists of HSPC (hydrogenated soya phosphatide)/Chol (SUV)/mPEG
2000(55:45:2, mol/mol), the liposome membrane material prescription that RPAKPAR modifies is HSPC/Chol/mPEG to-DOPE (methoxy poly (ethylene glycol) (molecular weight 2000)-dioleoyl phosphatidylethanolamine mixture)
2000-DOPE/ RPAKPAR-PEG
3350-DOPE (55:45:2:1, mol/mol).Take by weighing above-mentioned mould material respectively and be dissolved in chloroform, the decompression rotary evaporation is removed chloroform, gets even lipid film, vacuum-drying 24h.The 0.155M ammoniumsulphate soln that adds certain volume, 60 ℃ of water-bath concussion 2h get liposome turbid liquor.In 60 ℃ of water-baths, use high pressure homogenizer (if the liposome volume is less than 10mL and then uses the miniature device of extruding instead) successively liposome to be pushed 400,200,100 and the 50nm nucleopore membranes, blank liposome.With saline water is eluent, and the blank liposome wash-out is changed outer water through Sephadex G-50 gel column.Add Zorubicin physiological salt soln, 60 ℃ of water-bath 20min according to medicine fat than 1:10 (w/w).Through Sephadex G-50 gel column, remove free drug with the saline water wash-out, get Evacet.Use the dynamic light scattering determination particle diameter, referring to Fig. 4, liposome/FAM and RPAKPAR-liposome/FAM size distribution figure can be known by figure, both size and the no significant difference that distributes.Show that particle diameter all is about 70nm.
The checking of embodiment 9:RPAKPAR-liposome tumor cell in vitro target property
The monolayer culture PC-3 cell of taking the logarithm vegetative period with 0.25% trypsinase and 0.025% EDTA Disodium digestion single-layer culturing cell, is made into single cell suspension with the DMEM nutrient solution that contains 10% foetal calf serum, with every hole 1 * 10
5Individual cell inoculation is in 24 well culture plates, and every pore volume 1ml moves into culture plate in the CO2gas incubator, and 37 ℃, overnight cultures under 5% carbonic acid gas and the saturated humidity condition makes cell attachment.Next day, use the DMEM nutrient solution that contains 1% foetal calf serum to prepare the liposome/FAM and the RPAKPAR-liposome/FAM solution of a series of different concns.With the nutrient solution sucking-off in the culture plate, add the serial solution of liposome/FAM and RPAKPAR-liposome/FAM, hatch 1h for 37 ℃, inhale and abandon supernatant.Wash plate twice with PBS solution, observation of cell internalization situation under the fluorescence microscopy border.Referring to Fig. 5, RPAKPAR-liposome/FAM (A) and liposome/FAM (B) in 37 ℃ respectively with the fluorescence micrograph of PC-3 cytosis after 1 hour, can know by figure, the PC-3 cell to the intake of RPAKPAR-liposome/FAM much larger than liposome/FAM.The result shows that liposome/FAM can not be by the PC-3 cellular uptake, and RPAKPAR-liposome/FAM explains under the mediation of RPAKPAR that then by huge uptake the RPAKPAR-liposome has good external target property to the prostate cancer tumour cell.
Embodiment 10:RPAKPAR-liposome/DOX is to the growth in vitro restraining effect of PC-3 prostate cancer cell
Adopt mtt assay mensuration RPAKPAR-liposome/DOX and liposome/DOX growth in vitro restraining effect to tumour cell.The PC-3 cell that will be in logarithmic phase contains EDTA with 0.25% trypsin) digestion, cell counting, (corning, USA) every hole adds 200 μ l cell suspensions (2 * 10 in 96 orifice plates
3Cells), reserve three holes and add not celliferous nutrient solution as blank well, adherent 24h.With cell culture fluid liposome/DOX and RPAKPAR-liposome/DOX being diluted to DOX concentration is 6.25nM ~ 102.4 μ M, inhales and removes 96 orifice plate inner cells training liquid, and each hole adds the liposome soup of a series of concentration of 200 μ l.Each concentration is all established three multiple holes, reserves three holes that only add nutrient solution as control wells, cultivates 72h.With cell training liquid configuration MTT, concentration is 0.2mg/ml, inhales the soup that removes 96 orifice plates; Every hole adds MTT solution 200 μ l, cultivates 4h for 37 ℃, and careful the suction removed liquid; Every hole adds 150 μ l DMSO, and water-bath vibration 10min, ELIASA (Bio-TEK) detect absorbancy (detecting wavelength 570nm); Calculate cell survival rate with formula
Cell survival rate=(A
Experiment-A
Blank)/(A
Contrast-A
Blank)
And calculate the IC of liposome/DOX and the external anti-PC-3 cell of RPAKPAR-liposome/DOX according to survival rate
50Value is respectively 1.38 * 10
-4M and 1.66 * 10
-5M, the modification of RPAKPAR makes the IC of Evacet
50Value has reduced by 8.3 times, has significantly increased the external anti-prostate cancer cell growth effect of Evacet.
Embodiment 11:RPAKPAR-liposome/DOX to the prostate cancer tumor tissues body in the growth-inhibiting effect
To be in the PC-3 cell tryptase enzymic digestion of logarithmic phase, the PBS washing is scattered among the PBS, with 1 * 10
7Cell (100 μ l) concentration inoculated with subcutaneous injections is in shoulder blade position, nude mice right side, and the SPF environment is raised down.24 nude mices that formed the PC-3 Subcutaneous tumor are divided into 4 groups (6 every group) at random, are made as N.S. respectively, DOX, liposome/DOX and RPAKPAR-liposome/DOX group.100 μ l corresponding preparation were expelled in the nude mouse through foot pad tissue space after inoculation respectively on the the 15th, 20,25 day, and the total dose of Zorubicin is 10mg/kg.In inoculation back 30 days, nude mice is put to death, take out the Subcutaneous tumor piece, weigh and measurement volumes and weight.Volume is calculated by following formula:
V?(mm
3)?=?(d
2?×?D)?/?2
Wherein d and D are respectively the minor axis and the major diameter of lymphoglandula, and unit is mm.Referring to Fig. 6, the modification of RPAKPAR has significantly increased the growth in vitro inhibition effect of Evacet to PC-3 prostate cancer tumour cell, and result's demonstration is compared with liposome/DOX, and RPAKPAR-liposome/DOX significantly strengthens the tumor growth restraining effect.
Claims (10)
1. neuropil albumen-1 ligand polypeptide-polyoxyethylene glycol-phospholipid complex; It is characterized in that this mixture by neuropil albumen-1 ligand polypeptide, polyoxyethylene glycol and phosphatide three parts through the covalently bound linear block copolymers that forms; Wherein the mol ratio of neuropil albumen-1 ligand polypeptide, polyoxyethylene glycol and phosphatide is 1:1:1, and the aminoacid sequence of described neuropil albumen-1 ligand polypeptide is: RPAKPAR.
2. neuropil albumen according to claim 1-1 ligand polypeptide-polyoxyethylene glycol-phospholipid complex, the weight-average molecular weight that it is characterized in that described polyoxyethylene glycol is 400 ~ 8000.
3. neuropil albumen according to claim 2-1 ligand polypeptide-polyoxyethylene glycol-phospholipid complex is characterized in that described polyoxyethylene glycol weight-average molecular weight is 2000 ~ 5000.
4. neuropil albumen according to claim 1-1 ligand polypeptide-polyoxyethylene glycol-phospholipid complex is characterized in that described phosphatide is: DSPE, two palmityl phosphatidylethanolamines, DOPE, hydrogenated soya phosphatide acyl thanomin, hydrogenation egg phosphatide acyl thanomin, soybean phospholipid acyl thanomin or egg phosphatide acyl thanomin.
5. an active target liposomes carrier system contains with good grounds claim 1,2,3 or 4 described neuropil albumen-1 ligand polypeptide-polyoxyethylene glycol-phospholipid complex, it is characterized in that consisting of of this carrier system:
A. phosphatide;
B. SUV;
C. methoxy poly (ethylene glycol)-phospholipid complex;
D. neuropil albumen-1 ligand polypeptide-polyoxyethylene glycol-phospholipid complex;
Mol ratio between above-mentioned each composition is: a:b=5:1 ~ 1:5, a:c=1000:1 ~ 1000:100, a:d=1000:0.1 ~ 1000:100.
6. active target liposomes carrier system according to claim 5 is characterized in that the described phosphatide of component a is: egg phosphatide, hydrogenated soya phosphatide phatidylcholine, hydrogenation Yolk lecithin, two bay phosphatidyl cholines, dimyristoyl phosphatidyl choline, DPPC, DSPC, 1-mnyristoyl-2-palmitoylphosphatidyl choline, 1-palmityl-2-stearyl phosphatidylcholine, 1-stearyl-2-palmitoylphosphatidyl choline, 1-palmityl-2-oleoyl phosphatidylcholine, the inferior oleoyl phosphatidylcholine of 1-stearyl-2-, dioleoyl phospholipid phatidylcholine, hydrogenation DPPC, DSPC, two mnyristoyl phosphatidic acids, two mnyristoyl phosphatidic acids, two palmityl phosphatidic acids, two palmityl phosphatidic acids, G 12S3P, two mnyristoyl phosphatidylethanolamines, two palmityl phosphatidylethanolamines, kephalin acyl Serine, two mnyristoyl phosphatidylserines, two palmityl phosphatidylserines, egg phosphatide acyl glycerine, two lauryl POPGs, GLYCEROL,DIMYRISTOYL PHOSPHATIDYL, two palmityl POPGs, distearyl POPG, DOPG, brain sphingophospholipid, two palmityl sphingophospholipid or distearyl sphingophospholipid.
7. active target liposomes carrier system according to claim 5 is characterized in that the phosphatide in described methoxy poly (ethylene glycol)-phospholipid complex is: DSPE, two palmityl phosphatidylethanolamines, DOPE, hydrogenated soya phosphatide acyl thanomin, hydrogenation egg phosphatide acyl thanomin, soybean phospholipid acyl thanomin or egg phosphatide acyl thanomin.
8. active target liposomes carrier system according to claim 5 is characterized in that the methoxy poly (ethylene glycol) weight-average molecular weight in described methoxy poly (ethylene glycol)-phospholipid complex is 400 ~ 6000.
9. method for preparing according to claim 5,6,7 or 8 described active target liposomes carrier systems is characterized in that the concrete steps of this method are: take by weighing above-mentioned each component respectively and be dissolved in and prepare even lipid film, vacuum-drying in the chloroform; The ammoniumsulphate soln that adds 0.155M again, concussion obtains liposome turbid liquor in 20-70 ℃ of water-bath; In 20-70 ℃ of water-bath, pushed 400,200,100 and the 50nm nucleopore membranes more successively, blank liposome, be the active target liposomes carrier system that contains neuropil albumen-1 ligand polypeptide-polyoxyethylene glycol-phospholipid complex.
10. method according to claim 9 is characterized in that the preparing method's of described neuropil albumen-1 ligand polypeptide-polyoxyethylene glycol-phospholipid complex concrete steps are:
A. be that tertbutyloxycarbonyl-l-arginine (p-toluenesulfonyl)-acetparaminosalol benzyl ester Boc-Arg (Tos)-PAM resin of 0.6mmol/g is used N with substitution value, dinethylformamide DMF swelling is drained; Slough Boc protection base with trifluoroacetic acid TFA again, take out TFA;
B. use benzotriazole-N, N, N', DMF solution and the N of N'-tetramethyl-urea hexafluorophosphate HBTU, N-diisopropylethylamine DIEA activation tertbutyloxycarbonyl-L-Ala Boc-Ala gets the Boc-Ala activation solution;
C. with the Boc-Ala activation solution of step a gained resin with DMF washing back adding step b gained, 25 ℃ of joltings were reacted 25 minutes, took out dereaction liquid, and used the DMF washing resin;
D. repeating step a-c connects all the other amino acid in order by the CRPAKPAR sequence; Reaction finishes after scouring resin, TFA deprotection base, vacuum-drying;
E. the resin of steps d gained is put into polypeptide cutting pipe, add an amount of P-cresol, feed HF then, ice bath stirring reaction 1 hour; Reaction finishes the back decompression and takes out HF in the pipe, and raffinate is with an amount of ice ether sedimentation, crosses to filter deposition and with icing the ether washing precipitation; Deposition is crossed and is filtered filtrating again with the TFA dissolving; Filtrating is precipitated in the ice ether again, filters, and filter residue redissolves with water, and freeze-drying gets the pure article of CRPAKPAR;
F. the CRPAKPAR that step e is obtained is dissolved in the PBS solution of pH7.0; Get maleimide-polyoxyethylene glycol-phospholipid complex Mal-PEG-DSPE and be dissolved in DMF; Stirring reaction reacts completely to Mal-PEG-DSPE; Remove excessive CRPAKPAR and DMF, lyophilize obtains the neuropil albumen-1 ligand polypeptide-polyoxyethylene glycol-phospholipid complex of straight chain.
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