CN102091036A - Compound liposome containing anti-tumor drugs and preparation method and application thereof - Google Patents
Compound liposome containing anti-tumor drugs and preparation method and application thereof Download PDFInfo
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- CN102091036A CN102091036A CN2011100035702A CN201110003570A CN102091036A CN 102091036 A CN102091036 A CN 102091036A CN 2011100035702 A CN2011100035702 A CN 2011100035702A CN 201110003570 A CN201110003570 A CN 201110003570A CN 102091036 A CN102091036 A CN 102091036A
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Abstract
The invention belongs to a liposome, in particular relates to a compound liposome preparation with high targeting property of tumor tissues and efficient penetrability of tumor cells. The invention is characterized in that hyaluronic acid or sodium hyaluronate, tumor cell selective penetrating peptides and a liposome are used for together constructing a compound liposome. The compound liposome is characterized by utilizing the dual-tumor cell targeting property of the hyaluronic acid and tumor cell selective penetrating peptides to effectively solve the problem that because a conventional liposome and traditional cell penetrating peptides have no selectivity on normal cells and tumor cells, the toxicity is increased when the drug effect is increased in the therapeutic process, thereby fully meeting the clinical requirements of high efficiency and low toxicity when anti-tumor drugs are prepared into preparations for treating diseases.
Description
Technical field:
The invention belongs to medical technical field, use hyaluronic acid or hyaluronate sodium, tumor cell selectivity to wear film peptide and a kind of complex liposome of the common structure of liposome specifically.This complex liposome is characterised in that the dual tumor cell targeting that utilizes hyaluronic acid and tumor-selective to wear the film peptide, the difficult problem that toxicity also increased when drug effect increased when solving conventional liposome and tradition effectively and wearing the film peptide the normal and equal non-selectivity of tumor cell is caused its treatment, fully realize when tumour medicine is prepared into preparation and is used for the treatment of required efficiently, the clinical demand of low toxicity.
Background technology:
(cell-permeable peptides CPP) is the polypeptide that a class has strong cell membrane penetration capacity, and they have efficiently passes cell membrane and the characteristics of damaging cells membrane structure and function not to wear the film peptide.To wear the film peptide and carry various molecules or nano-carrier permeate through cell membranes, and penetrate and brought into play huge effect aspect the various cell membrane at mediated gene, protein, polypeptide, micromolecule, nano-particle and liposome etc. by modes such as covalent bond or non-covalent bond couplings.
Existing studies show that, traditional film peptide and conventional liposome (not modified) worn be to normal structure and the general non-selectivity of tumor tissues, even initiatively the targeting liposome also has the distribution of some in normal structure, if with traditional film peptide such as R of wearing
8After being used for modifying conventional liposome, because that wears the film peptide efficiently wears membrane property, in the medication amount, must follow the corresponding increase of normal cell Chinese medicine amount in increasing tumor cell, this will cause the more major injury (toxic and side effects obviously increases) of normal cell tissue.Therefore, a kind ofly have tumor cell and optionally wear the film peptide if can make up, compare with the common film peptide of wearing of employing, it is combined and carry it with nano-carriers such as liposomees by modes such as covalent bond or non-covalent bond couplings pass cell membrane, will have bigger application prospect in the field of targeted therapy tumor disease.
The tumor cell selectivity is worn film peptide (selective cell penetration peptides on tumor cell, SCPPt) wear a kind of novel cell-penetrating peptide that innovation research goes out on the film peptide basis for this laboratory at classics, it has has higher penetrating efficient to tumor cell membrane, do not penetrate or penetrate the lower characteristics of efficient and human cell membrane almost had, mainly form by 5～12 arginine and 3 histidine.Have this laboratory invention aminoacid sequence wear the film peptide under normal pH environment, only have small positive charge, can't combine with the cell surface height and wear film; And the environment owing to its slant acidity makes histidine and collaborative the showing of arginine have a large amount of positive charges when arriving the tumor tissue cell surface, thereby can be efficiently combine, embody the stronger effect that penetrates tumor cell membrane with electronegative cell surface.
Hyaluronic acid (HA) is to be the straight chain polymer polysaccharide of disaccharidase unit's composition with D-glucuronic acid-N-acetylglucosamine, it is a kind of anionic species that extensively is present in extracellular matrix, cell surface and the cell interior of the multiple tissue of body, belong to endogenous material in the body, do not bring out immunoreation, safe.Recent study shows, HA can with the receptors bind such as CD44, RHAMM of tumor cell surface high expressed, activation is at the intracellular signaling pathway of HA or activate the internalization of HA, and regulates the behaviors such as motion of cell, and can be by the HA enzyme that the tumor cell surface height the is expressed elimination of degrading rapidly.In addition, high concentration HA also helps macrophage migration in the lymph node, and the effect with killing tumor cell is engulfed in performance.According to the literature, HA can hold macrophage by its viscoelasticity macromolecular network structure that effect forms, isolates with macrophage and by phagocyte or granule, thus the phagocytosis of restriction macrophage, prolong the blood circulation time of its bag suppressed by vector, have long cyclical effect.HA is an electronegativity, and at electropositive surface of liposome, and proof can significantly improve the tumor-targeting of liposome by the electrostatic interaction self assembly.In view of HA has excellent biological compatibility and degradability, long circulation and elecrtonegativity, non-immunogenicity, tumor cell high-affinity and the effect of antitumor auxiliary treatment etc., become one of research focus of present antitumor active targeting vector.
Summary of the invention:
Goal of the invention
Purpose of the present invention aims to provide complex liposome of a kind of tumor tissues height targeting, the efficient penetrance of tumor cell and its production and use.
Technical scheme
At the foregoing invention purpose, the invention provides following technical scheme:
A kind of complex liposome that contains antitumor drug is characterized in that: it contains antitumor drug, lipid, hyaluronic acid or hyaluronate sodium, the tumor cell selectivity is worn the film peptide.
Described complex liposome is characterized in that: press mass ratio, it contains the antitumor drug of 0.01%-30%, the lipid of 1%-98%, the 0.001%-20% hyaluronate sodium, the tumor cell selectivity of 0.001%-20% is worn the film peptide, the alcohol of 0-20%, the antioxidant of 0-20%, the antiseptic of 0-20%, the pH regulator agent of 0-20%, the phase transformation regulator of 0-20%, the long recycled material of 0-20%, the isoosmotic adjusting agent of 0-20%, the lyophilizing proppant of 0-95%, the water of 0-95%.
Described complex liposome is characterized in that described antitumor drug is selected from one or more in the following medicine: paclitaxel, Docetaxel, actinomycin D, doxorubicin, epirubicin, daunorubicin, vincristine, vinorelbine, cisplatin, oxaliplatin, methotrexate, fluorouracil, mercaptopurine, cytosine arabinoside, doxifluridine, etoposide, teniposide, camptothecine, hydroxy camptothecin, topotecan, irinotecan, mitoxantrone, cyclophosphamide, ifosfamide, ametycin, busulfan, lomustine, carmustine, semustine, nimustine, Ranimustine, gemcitabine, capecitabine, decitabine, ancitabine, cisplatin, bleomycin, Bleomycin A5, gamlogic acid, the various pharmaceutical salts of neogambogic acid and these medicines.
Described complex liposome is characterized in that described lipid is phospholipid and cholesterol, and phospholipid is selected from one or more in natural phospholipid, semi-synthetic phospholipid and the complete synthesis phospholipid.
Described complex liposome, the relative molecular mass that it is characterized in that described hyaluronic acid or hyaluronate sodium is from 4 * 10
3Dalton to 2 * 10
7Dalton.
Described complex liposome is characterized in that described tumor cell selectivity wears the film peptide and for tumor cell or tissue stronger cell membrane penetration is arranged, and maybe can not see through the cell membrane penetration of normal cell or tissue is more weak.
Described complex liposome is characterized in that described tumor cell selectivity wears the film peptide and be selected from the following polypeptide one or more: RRRRRHHH (R
5H
3, R is at C end or N end), RRRRRRHHH (R
6H
3, R is at C end or N end), RRRRRRRHHH (R
7H
3, R is at C end or N end), RRRRRRRRHHH (R
8H
3, R is at C end or N end), RRRRRRRRRHHH (R
9H
3, R is at C end or N end), RRRRRRRRRRHHH (R
10H
3, R is at C end or N end), RRRRRRRRRRRHHH (R
11H
3, R is at C end or N end), RRRRRRRRRRRRHHH (R
12H
3, R is at C end or N end), their N end is by fatty acid modifying, and the preferred carbochain of fatty acid contains 8-18 carbon atom, more preferably stearic acid.
Described complex liposome is characterized in that preparing the pharmaceutical preparation for the treatment of tumor.
Useful achievement
After this complex liposome injects vein, with long cyclicity, tumor-targeting and the biological degradability that is at first had by outer hyaluronic acid bag, initiatively be gathered in tumor tissues and cell surface and degraded rapidly by this position high concentration hyaluronidase, make the tumor cell selectivity wear the film peptide and be exposed to surface of liposome, give full play to its efficient penetration to tumor cell, carry liposome and enter tumor cell more, thus oncocyte and bring into play cytotoxicity when the antitumor drug that liposome is sealed enters more.This complex liposome is when solving a tumor tissues medicine wellability difference difficult problem, effectively reduce cell toxicity medicament to Normocellular injury, significantly improve cell toxicity medicament commonly used and wear the film peptide, have boundless application prospect owing to the toxic and side effects that the negative for tumor cells targeting causes.
The specific embodiment:
R in the following example is an arginine, and H is a histidine
Embodiment 1
Wear film peptide R with hyaluronate sodium, the tumor cell selectivity that reaches of the present invention
8H
3With Doxil the preparation method of this complex liposome is described.
Add 1.39gRinkamide-MBHA resin (0.74mmol/g) in 50mL side band arm, arm have the round-bottomed flask of sand plate filter element, add 20mL DMF swelling 10 minutes, sucking filtration removes solvent.Add 20mL 20% piperidines/DMF solution then, stirred sucking filtration 30 minutes.With DMF washing resin 6 times, sucking filtration removes solvent.(Pbf is 2 to add 2.00g (3.09mmol) Fmoc-Arg (Pbf)-OH in reaction bulb; 2; 4; 6; 7-pentamethyl Dihydrobenzofuranes-5-sulfonyl), 0.64g (3.09mmol) dicyclohexylcarbodiimide and 0.42g (3.09mmol) HOBt (I-hydroxybenzotriazole) and 20mLDMF, stir under the room temperature and carry out condensation reaction.React the resin that takes a morsel after 2 hours and carry out the 1,2,3-indantrione monohydrate chromogenic reaction, the result shows that condensation reaction is complete.Sucking filtration is used DMF washing resin 6 times, and sucking filtration removes solvent.(protection) aminoacid of C end is connected on the resin.Reactions steps repeats top-operation to second of being connected with then that C held in back to last (protection) aminoacid, promptly from 20% piperidines/DMF solution deprotection, after the 1,2,3-indantrione monohydrate colour test with the DMF washing only.If it is incomplete that the 1,2,3-indantrione monohydrate colour test shows condensation, then can prolong the condensation time until fully.All with the Fmoc protection, aminoacid is followed successively by all amino acid whose a-amino: Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-His (Trt)-OH, Fmoc-His (Trt)-OH, Fmoc-His (Trt)-OH.Take off the Fmoc protection at last, drop into stearic acid and N end amino and finish condensation reaction.Polypeptide resin is washed for the third time with methanol, vacuum drying.
The above-mentioned resin that has connect peptide is joined in the 50mL round-bottomed flask, add 15mL reagent K (TFA/ thioanisole/EDT/ phenol/water=87.5/5.5/2.5/2.5/2.5 (v/v)), stirred 3 hours under the room temperature.Filter, collect filtrate.With give a baby a bath on the third day after its birth time resin of trifluoroacetic acid.Merging filtrate revolves steaming with filtrate and removes most trifluoroacetic acid, and adding volume is that 8-10 ether sedimentation doubly is connected to stearic polypeptide, puts into refrigerator overnight.Centrifugal, remove ether, vacuum drying obtains the crude product that N holds the RRRRRRRRHHH that is modified by stearic acid.
The product that 100mg is above-mentioned is dissolved in the 2mL pure water, uses preparation reversed-phase HPLC purification, collects to keep main peak, collects liquid and contracts through revolving inspissation, and lyophilization gets the pure product that N holds the RRRRRRRRHHH that is modified by stearic acid then.Mass spectrum shows that its molecular weight is 2143, conforms to value of calculation.The final N of acquisition holds the tumor cell selectivity of being modified by stearic acid to wear film peptide R
8H
3
Take by weighing injection soybean phospholipid and cholesterol in eggplant type bottle according to recipe quantity, add a certain amount of chloroform it is fully dissolved, and it is some to add bead.Vacuum rotary steam 1 hour is waved except that organic solvent formation medicine membrane of lipoprotein.Finish back continuation evacuation and spend the night, to remove trace organic solvents.Add the hand aquation medicine of citric acid solution (pH4.0) room temperature membrane of lipoprotein, till cleaning to eggplant type bottle wall, sample is uniform emulsion.Probe is ultrasonic to reduce particle diameter in the ice-water bath.Centrifugal 3000rpm is to remove the ultrasonic metal fillings that may drop of probe.Get supernatant and cross 0.45 μ m respectively, 0.22 μ m polycarbonate leaching film respectively carries out granulate 5 times can make blank liposome.In the liposome dispersion, add doxorubicin hydrochloride then, and with the Na of 1mol/L
2HPO
4PH value is adjusted to 7.0.Adjust volume at last, making doxorubicin concentration is 1mg/mL.This dispersion is hatched 20min in 55 ℃ promptly get Doxil.
Precision takes by weighing 8mg N and holds the tumor cell selectivity of being modified by stearic acid to wear film peptide R
8H
3, and it fully is dissolved in the distilled water of 2mL.Under room temperature, this solution slowly is added drop-wise in the above Doxil dispersion that makes, the dropping process keeps magnetic agitation constantly.Drip to finish the back and continue to stir 30min, then this dispersion is hatched at 4 ℃ and can make the tumor cell selectivity in 8 hours and wear film peptide R
8H
3The Doxil of modifying.The envelop rate of liposome before and after modifying and the investigation result of stability are shown that the tumor cell selectivity is worn film peptide R
8H
3Modification to Doxil does not produce appreciable impact to the physicochemical property of common Doxil, and envelop rate is greater than 90%, and room temperature is placed 24 hours percolation ratios less than 4%.
It is 3 * 10 that precision takes by weighing relative molecular mass
5Hyaluronate sodium 5mg, be dissolved in the distilled water of 4mL, regulate pH value to 6.0.Under room temperature, this solution slowly is added drop-wise to the tumor cell selectivity that makes and wears film peptide R
8H
3In the dispersion of the Doxil of modifying, the dropping process keeps magnetic agitation constantly.Continuation stirring 30min promptly was able to hyaluronate sodium after dropping finished, the tumor cell selectivity is worn film peptide R
8H
3The complex liposome constructed with Doxil.This complex liposome mean diameter is 147.8nm, and the zeta current potential is-20.3mV, and polydispersity index PDI is 0.236, and the doxorubicin hydrochloride envelop rate is 92.7%.Sucrose with 1% is freeze drying protectant, above-mentioned complex liposome is prepared into lyophilized powder preserves, and the glucose solution with 5% during use redissolves for the injection use.
Embodiment 2
Wear film peptide R with hyaluronate sodium, the tumor cell selectivity that reaches of the present invention
8H
3With the vincristine sulfate liposome preparation method of this complex liposome is described.
Add 1.39gRinkamide-MBHA resin (0.74mmol/g) in 50mL side band arm, arm have the round-bottomed flask of sand plate filter element, add 20mL DMF swelling 10 minutes, sucking filtration removes solvent.Add 20mL 20% piperidines/DMF solution then, stirred sucking filtration 30 minutes.With DMF washing resin 6 times, sucking filtration removes solvent.(Pbf is 2 to add 2.00g (3.09mmol) Fmoc-Arg (Pbf)-OH in reaction bulb; 2; 4; 6; 7-pentamethyl Dihydrobenzofuranes-5-sulfonyl), 0.64g (3.09mmol) dicyclohexylcarbodiimide and 0.42g (3.09mmol) HOBt (I-hydroxybenzotriazole) and 20mLDMF, stir under the room temperature and carry out condensation reaction.React the resin that takes a morsel after 2 hours and carry out the 1,2,3-indantrione monohydrate chromogenic reaction, the result shows that condensation reaction is complete.Sucking filtration is used DMF washing resin 6 times, and sucking filtration removes solvent.(protection) aminoacid of C end is connected on the resin.Reactions steps repeats top-operation to second of being connected with then that C held in back to last (protection) aminoacid, promptly from 20% piperidines/DMF solution deprotection, after the 1,2,3-indantrione monohydrate colour test with the DMF washing only.If it is incomplete that the 1,2,3-indantrione monohydrate colour test shows condensation, then can prolong the condensation time until fully.All with the Fmoc protection, aminoacid is followed successively by all amino acid whose a-amino: Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-His (Trt)-OH, Fmoc-His (Trt)-OH, Fmoc-His (Trt)-OH.Take off the Fmoc protection at last, drop into stearic acid and N end amino and finish condensation reaction.Polypeptide resin is washed for the third time with methanol, vacuum drying.
The above-mentioned resin that has connect peptide is joined in the 50mL round-bottomed flask, add 15mL reagent K (TFA/ thioanisole/EDT/ phenol/water=87.5/5.5/2.5/2.5/2.5 (v/v)), stirred 3 hours under the room temperature.Filter, collect filtrate.With give a baby a bath on the third day after its birth time resin of trifluoroacetic acid.Merging filtrate revolves steaming with filtrate and removes most trifluoroacetic acid, and adding volume is that 8-10 ether sedimentation doubly is connected to stearic polypeptide, puts into refrigerator overnight.Centrifugal, remove ether, vacuum drying obtains the crude product that N holds the RRRRRRRRHHH that is modified by stearic acid.
The product that 100mg is above-mentioned is dissolved in the 2mL pure water, uses preparation reversed-phase HPLC purification, collects to keep main peak, collects liquid and contracts through revolving inspissation, and lyophilization gets the pure product that N holds the RRRRRRRRHHH that is modified by stearic acid then.Mass spectrum shows that its molecular weight is 2143, conforms to value of calculation.The final N of acquisition holds the tumor cell selectivity of being modified by stearic acid to wear film peptide R
8H
3
Take by weighing hydrogenated soya phosphatide and cholesterol according to recipe quantity, be dissolved in an amount of dichloromethane: in the methanol (volume ratio 1: 1), wave except that organic solvent in 50 ℃ of water-baths.Vacuum rotary steam 1 hour is waved except that organic solvent formation medicine membrane of lipoprotein.Finish back continuation evacuation and spend the night, to remove trace organic solvents.Add 50 ℃ of water-bath rotations of citric acid soln (pH4.3) and wash film, till cleaning to eggplant type bottle wall, sample is uniform emulsion.Probe is ultrasonic to reduce particle diameter in the ice-water bath.Centrifugal 3000rpm is to remove the ultrasonic metal fillings that may drop of probe.Get supernatant and cross 0.45 μ m respectively, 0.22 μ m polycarbonate leaching film respectively carries out granulate 3 times can make blank liposome.The Na that in the liposome dispersion, adds vincristine sulfate solution and pH9.0 then
2HPO
4Solution.Add water for injection and make outer water regulate pH to 7.3, promptly get the vincristine sulfate liposome at 55 ℃ of water bath heat preservation 15min.
Precision takes by weighing 8mg N and holds the tumor cell selectivity of being modified by stearic acid to wear film peptide R
8H
3, and it fully is dissolved in the distilled water of 4mL.Under room temperature, this solution slowly is added drop-wise in the above vincristine sulfate star liposome dispersion that makes, the dropping process keeps magnetic agitation constantly.Drip to finish the back and continue to stir 30min, then this dispersion is hatched at 4 ℃ and can make the tumor cell selectivity in 8 hours and wear film peptide R
8H
3The vincristine sulfate liposome of modifying.The envelop rate of liposome before and after modifying and the investigation result of stability are shown that the tumor cell selectivity is worn film peptide R
8H
3Modification to the vincristine sulfate liposome does not produce appreciable impact to the physicochemical property of common vincristine sulfate liposome, and envelop rate is greater than 80%, and room temperature is placed 24 hours percolation ratios less than 4%.
It is 3 * 10 that precision takes by weighing relative molecular mass
5Hyaluronate sodium 8mg, be dissolved in the distilled water of 4mL, regulate pH value to 6.0.Under room temperature, this solution slowly is added drop-wise to the tumor cell selectivity that makes and wears film peptide R
8H
3In the dispersion of the vincristine sulfate liposome of modifying, the dropping process keeps magnetic agitation constantly.Continuation stirring 30min promptly was able to hyaluronate sodium after dropping finished, the tumor cell selectivity is worn film peptide R
8H
3The complex liposome constructed with the vincristine sulfate liposome.This complex liposome mean diameter is 203.8nm, and the zeta current potential is-23.7mV, and polydispersity index PDI is 0.156, and the vincristine sulfate envelop rate is 86.1%.Sucrose and mannitol with certain proportioning are freeze drying protectant, above-mentioned complex liposome is prepared into lyophilized powder preserves, and the glucose solution with 5% during use redissolves for the injection use.
Embodiment 3
Wear film peptide R with hyaluronate sodium, the tumor cell selectivity that reaches of the present invention
10H
3With the methotrexate liposome preparation method of this complex liposome is described.
Add 1.65gRinkamide-MBHA resin (0.74mmol/g) in 50mL side band arm, arm have the round-bottomed flask of sand plate filter element, add 20mL DMF swelling 10 minutes, sucking filtration removes solvent.Add 20mL 20% piperidines/DMF solution then, stirred sucking filtration 30 minutes.With DMF washing resin 6 times, sucking filtration removes solvent.(Pbf is 2 to add 2.38g (3.67mmol) Fmoc-Arg (Pbf)-OH in reaction bulb; 2; 4; 6; 7-pentamethyl Dihydrobenzofuranes-5-sulfonyl), 0.76g (3.67mmol) dicyclohexylcarbodiimide and 0.50g (3.67mmol) HOBt (I-hydroxybenzotriazole) and 20mLDMF, stir under the room temperature and carry out condensation reaction.React the resin that takes a morsel after 2 hours and carry out the 1,2,3-indantrione monohydrate chromogenic reaction, the result shows that condensation reaction is complete.Sucking filtration is used DMF washing resin 6 times, and sucking filtration removes solvent.(protection) aminoacid of C end is connected on the resin.Reactions steps repeats top-operation to second of being connected with then that C held in back to last (protection) aminoacid, promptly from 20% piperidines/DMF solution deprotection, after the 1,2,3-indantrione monohydrate colour test with the DMF washing only.If it is incomplete that the 1,2,3-indantrione monohydrate colour test shows condensation, then can prolong the condensation time until fully.All with the Fmoc protection, aminoacid is followed successively by all amino acid whose a-amino: Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-His (Trt)-OH, Fmoc-His (Trt)-OH, Fmoc-His (Trt)-OH.Take off the Fmoc protection at last, drop into stearic acid and N end amino and finish condensation reaction.Polypeptide resin is washed for the third time with methanol, vacuum drying.
The above-mentioned resin that has connect peptide is joined in the 50mL round-bottomed flask, add 15mL reagent K (TFA/ thioanisole/EDT/ phenol/water=87.5/5.5/2.5/2.5/2.5 (v/v)), stirred 3 hours under the room temperature.Filter, collect filtrate.With give a baby a bath on the third day after its birth time resin of trifluoroacetic acid.Merging filtrate revolves steaming with filtrate and removes most trifluoroacetic acid, and adding volume is that 8-10 ether sedimentation doubly is connected to stearic polypeptide, puts into refrigerator overnight.Centrifugal, remove ether, vacuum drying obtains the crude product that N holds the RRRRRRRRRRHHH that is modified by stearic acid.
The above-mentioned product of 100mg is dissolved in the 2mL pure water, uses preparation reversed-phase HPLC purification, collect the reservation main peak, collect liquid and contract through revolving inspissation, lyophilization gets the pure product that N holds the RRRRRRRRRRHHH that is modified by stearic acid then.The mass spectrum of pure product shows that its molecular weight is 2491, conforms to value of calculation.The final N of acquisition holds the tumor cell selectivity of being modified by stearic acid to wear film peptide R
10H
3
Take by weighing Ovum Gallus domesticus Flavus lecithin and cholesterol in eggplant type bottle according to recipe quantity, add the 4mL ether dissolution, drip methotrexate aqueous solution 2mL while in ice-water bath, stir.Behind the uniform and stable w/o type emulsion to be formed, boil off ether, obtain brown emulsion in 40 ℃ of vacuum rotary steams, the ultrasonic 15min of 100W power probe, last 0.45 μ m Merlon microporous filter membrane promptly gets the methotrexate liposome 5 times.Methotrexate liposome dispersion is packed in the bag filter, is the dialysis medium with the distilled water, and not entrapped drug was removed in dialysis in 24 hours under the room temperature.
Precision takes by weighing 10mg N and holds the tumor cell selectivity of being modified by stearic acid to wear film peptide R
10H
3, and it fully is dissolved in the distilled water of 4mL.Under room temperature, this solution slowly is added drop-wise in the above methotrexate liposome dispersion that makes, the dropping process keeps magnetic agitation constantly.Drip to finish the back and continue to stir 30min, then this dispersion is hatched at 4 ℃ and can make the tumor cell selectivity in 8 hours and wear film peptide R
10H
3The methotrexate liposome of modifying.The envelop rate of liposome before and after modifying and the investigation result of stability are shown that the tumor cell selectivity is worn film peptide R
10H
3Modification to the methotrexate liposome does not produce appreciable impact to the physicochemical property of common methotrexate liposome, and envelop rate is greater than 70%, and room temperature is placed 24 hours percolation ratios less than 5%.
It is 1 * 10 that precision takes by weighing relative molecular mass
5Hyaluronate sodium 8mg, be dissolved in the distilled water of 4mL, regulate pH value to 6.0.Under room temperature, this solution slowly is added drop-wise to the tumor cell selectivity that makes and wears film peptide R
10H
3In the dispersion of the methotrexate liposome of modifying, the dropping process keeps magnetic agitation constantly.Continuation stirring 30min promptly was able to hyaluronate sodium after dropping finished, the tumor cell selectivity is worn film peptide R
10H
3The complex liposome constructed with the methotrexate liposome.This complex liposome mean diameter is 276.4nm, and the zeta current potential is-24.3mV, and polydispersity index PDI is 0.186, and the methotrexate envelop rate is 72.3%.Sucrose with 1% is freeze drying protectant, above-mentioned complex liposome is prepared into lyophilized powder preserves, and the glucose solution with 5% during use redissolves for the injection use.
Embodiment 4
Wear film peptide R with hyaluronate sodium, the tumor cell selectivity that reaches of the present invention
9H
3With the neogambogic acid liposome preparation method of this complex liposome is described.
Add 1.52gRinkamide-MBHA resin (0.74mmol/g) in 50mL side band arm, arm have the round-bottomed flask of sand plate filter element, added the 20mLDMF swelling 10 minutes, sucking filtration removes solvent.Add 20mL 20% piperidines/DMF solution then, stirred sucking filtration 30 minutes.With DMF washing resin 6 times, sucking filtration removes solvent.(Pbf is 2 to add 2.19g (3.38mmol) Fmoc-Arg (Pbf)-OH in reaction bulb; 2; 4; 6; 7-pentamethyl Dihydrobenzofuranes-5-sulfonyl), 0.70g (3.38mmol) dicyclohexylcarbodiimide and 0.46g (3.38mmol) HOBt (I-hydroxybenzotriazole) and 20mLDMF, stir under the room temperature and carry out condensation reaction.React the resin that takes a morsel after 2 hours and carry out the 1,2,3-indantrione monohydrate chromogenic reaction, the result shows that condensation reaction is complete.Sucking filtration is used DMF washing resin 6 times, and sucking filtration removes solvent.(protection) aminoacid of C end is connected on the resin.Reactions steps repeats top-operation to second of being connected with then that C held in back to last (protection) aminoacid, promptly from 20% piperidines/DMF solution deprotection, after the 1,2,3-indantrione monohydrate colour test with the DMF washing only.If it is incomplete that the 1,2,3-indantrione monohydrate colour test shows condensation, then can prolong the condensation time until fully.All with the Fmoc protection, aminoacid is followed successively by all amino acid whose a-amino: Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-His (Trt)-OH, Fmoc-His (Trt)-OH, Fmoc-His (Trt)-OH.Take off the Fmoc protection at last, drop into stearic acid and N end amino and finish condensation reaction.Polypeptide resin is washed for the third time with methanol, vacuum drying.
The above-mentioned resin that has connect peptide is joined in the 50mL round-bottomed flask, add 15mL reagent K (TFA/ thioanisole/EDT/ phenol/water=87.5/5.5/2.5/2.5/2.5 (v/v)), stirred 3 hours under the room temperature.Filter, collect filtrate.With give a baby a bath on the third day after its birth time resin of trifluoroacetic acid.Merging filtrate revolves steaming with filtrate and removes most trifluoroacetic acid, and adding volume is that 8-10 ether sedimentation doubly is connected to stearic polypeptide, puts into refrigerator overnight.Centrifugal, remove ether, vacuum drying obtains the crude product that N holds the RRRRRRRRRHHH that is modified by stearic acid.
The above-mentioned product of 100mg is dissolved in the 2mL pure water, uses preparation reversed-phase HPLC purification, collect the reservation main peak, collect liquid and contract through revolving inspissation, lyophilization gets the pure product that N holds the RRRRRRRRRHHH that is modified by stearic acid then.The mass spectrum of pure product shows that its molecular weight is 2317, conforms to value of calculation.The final N of acquisition holds the tumor cell selectivity of being modified by stearic acid to wear film peptide R
9H
3
Take by weighing soybean phospholipid, cholesterol and neogambogic acid according to recipe quantity, be dissolved in altogether in an amount of chloroform.This solution is placed 100mL eggplant type bottle, and 40 ℃ of water bath with thermostatic control decompression rotary evaporations extract chloroform, promptly form uniform medicine membrane of lipoprotein at the bottle wall after 1 hour.Finish back continuation evacuation and spend the night, to remove trace organic solvents.The PBS buffer solution that adds pH7.4, the normal pressure rotation is washed film to thin film and is come off fully under the room temperature.Adopt the ultrasonic 15min of probe Ultrasound Instrument to reduce particle diameter, 3000rpm is centrifugal to remove the ultrasonic metal fillings that may drop of probe then.Get supernatant and cross 0.45 μ m respectively, each once can make the neogambogic acid liposome 0.22 μ m polycarbonate leaching film.
Precision takes by weighing 10mg N and holds the tumor cell selectivity of being modified by stearic acid to wear film peptide R
9H
3, and it fully is dissolved in the distilled water of 4mL.Under room temperature, this solution slowly is added drop-wise in the above neogambogic acid liposome dispersion that makes, the dropping process keeps magnetic agitation constantly.Drip to finish the back and continue to stir 30min, then this dispersion is hatched at 4 ℃ and can make the tumor cell selectivity in 8 hours and wear film peptide R
9H
3The neogambogic acid liposome of modifying.The envelop rate of liposome before and after modifying and the investigation result of stability are shown that the tumor cell selectivity is worn film peptide R
9H
3Modification to the neogambogic acid liposome does not produce appreciable impact to the physicochemical property of common neogambogic acid liposome, and envelop rate is greater than 80%, and room temperature is placed 24 hours percolation ratios less than 3%.
It is 5 * 10 that precision takes by weighing relative molecular mass
4Hyaluronate sodium 8mg, be dissolved in the distilled water of 4mL, regulate pH value to 6.0.Under room temperature, this solution slowly is added drop-wise to the tumor cell selectivity that makes and wears film peptide R
9H
3In the dispersion of the neogambogic acid liposome of modifying, the dropping process keeps magnetic agitation constantly.Continuation stirring 30min promptly was able to hyaluronate sodium after dropping finished, the tumor cell selectivity is worn film peptide R
9H
3The complex liposome constructed with the neogambogic acid liposome.This complex liposome mean diameter is 147.6nm, and the zeta current potential is-19.3mV, and polydispersity index PDI is 0.206, and the neogambogic acid envelop rate is 82.3%.Mannitol with 1% is freeze drying protectant, above-mentioned complex liposome is prepared into lyophilized powder preserves, and redissolves for the injection use with 0.9% sodium chloride solution during use.
Embodiment 5
Wear film peptide (R with the tumor cell selectivity
5H
3, R
6H
3, R
7H
3, R
8H
3, R
9H
3, R
10H
3, R
11H
3, R
12H
3, R is at N end or C end) and externally press down the tumor experiment illustrates this preparation for example antitumor efficacy with the constructed tumor cell selectivity of vincristine sulfate liposome is worn vincristine sulfate liposome that the film peptide modifies.
Take by weighing hydrogenated soya phosphatide and cholesterol according to recipe quantity, be dissolved in an amount of dichloromethane: in the methanol (volume ratio 1: 1), wave except that organic solvent in 50 ℃ of water-baths.Vacuum rotary steam 1 hour is waved except that organic solvent formation medicine membrane of lipoprotein.Finish back continuation evacuation and spend the night, to remove trace organic solvents.Add 50 ℃ of water-bath rotations of citric acid soln (pH4.3) and wash film, till cleaning to eggplant type bottle wall, sample is uniform emulsion.Probe is ultrasonic to reduce particle diameter in the ice-water bath.Centrifugal 3000rpm is to remove the ultrasonic metal fillings that may drop of probe.Get supernatant and cross 0.45 μ m respectively, 0.22 μ m polycarbonate leaching film respectively carries out granulate 3 times can make blank liposome.The Na that in the liposome dispersion, adds vincristine sulfate solution and pH9.0 then
2HPO
4Solution.Add water for injection and make outer water regulate pH to 7.3, promptly get the vincristine sulfate liposome at 55 ℃ of water bath heat preservation 15min.
Precision takes by weighing 8mg N and holds the tumor cell selectivity of being modified by stearic acid to wear film peptide R
5H
3, and it fully is dissolved in the distilled water of 4mL.Under room temperature, this solution slowly is added drop-wise in the above vincristine sulfate star liposome dispersion that makes, the dropping process keeps magnetic agitation constantly.Drip to finish the back and continue to stir 30min, then this dispersion is hatched at 4 ℃ and can make the tumor cell selectivity in 8 hours and wear film peptide R
8H
3The vincristine sulfate liposome of modifying.The envelop rate of liposome before and after modifying and the investigation result of stability are shown that the tumor cell selectivity is worn film peptide R
8H
3Modification to the vincristine sulfate liposome does not produce appreciable impact to the physicochemical property of common vincristine sulfate liposome, and envelop rate is greater than 80%, and room temperature is placed 24 hours percolation ratios less than 4%.
Other tumor cell selectivitys are worn film peptide (R
6H
3, R
7H
3, R
8H
3, R
9H
3, R
10H
3, R
11H
3, R
12H
3, R is at N end or C end) and can adopt identical method to prepare with the constructed liposome dosage form of vincristine sulfate liposome.
In addition, adopt identical method to prepare the vincristine sulfate liposome, and worn film peptide R with N end by what stearic acid was modified as stated above
8Preparation R
8The vincristine sulfate liposome of modifying.
Selection is in the human hepatoma cell strain HepG2 of exponential phase, discards culture medium and adds trypsinization 1min, adds an amount of DMEM culture medium and stops digestion, and microscopy calculates cell density, adds a certain amount of culture medium and makes 5 * 10
4The cell suspension of individual/mL.Add cell suspension to 96 orifice plate, every hole 200 μ L.37 ℃, 5%CO
2Incubator was cultivated 24 hours.Prepare various tumor cell selectivitys and wear vincristine sulfate liposome, the R that the film peptide is modified
8The medicinal liquid of the variable concentrations of the vincristine sulfate liposome of modifying, the vincristine sulfate liposome of unmodified and vincristine sulphate for injection.96 orifice plates discard culture medium, adding the drug solution of 200 μ L with the dilution of DMEM culture medium, vincristine sulfate final concentration onboard is: 1,3,10,15,30,60,120,180,240,300 μ g/mL, each concentration is established 3 multiple holes, is contrast with not dosing group.Move into 37 ℃ after the dosing, 5%CO
2Incubator was cultivated 48 hours.Then, discard liquid in the hole, add DMEM culture medium 190 μ L/ holes and MTT (5mg/mL) 10 μ L/ holes.37 ℃, 5%CO
2After hatching 4 hours in the incubator, discard original liquid in each hole, add dimethyl sulfoxide 200 μ L/ holes with dissolution precipitation.Under the 570nm wavelength, measure each hole optical density value with microplate reader behind the 30min.Calculate the medicine suppression ratio: medicine suppression ratio=(1-experimental group optical density value/matched group optical density value) * 100%.The result as shown in Table 1, with R
8The vincristine sulfate liposome of modifying, the vincristine sulfate liposome of unmodified are compared with vincristine sulphate for injection, and the cytotoxicity that various tumor cell selectivitys are worn the vincristine sulfate liposome of film peptide modification obviously strengthens (P＜0.05).
Each preparation of table one experimental group is to the half-inhibition concentration (IC of human hepatoma cell strain HepG2
50)
Preparation IC
50(μ g/mL)
Vincristine sulphate for injection 46.3 ± 2.7
The vincristine sulfate liposome 36.1 ± 3.0 of unmodified
R
8The vincristine sulfate liposome of modifying 30.8 ± 1.9
R
5H
3The vincristine sulfate liposome of modifying 25.3 ± 2.7
R
6H
3The vincristine sulfate liposome of modifying 23.3 ± 2.3
R
7H
3The vincristine sulfate liposome of modifying 21.2 ± 2.1
R
8H
3The vincristine sulfate liposome of modifying 19.8 ± 2.4
R
9H
3The vincristine sulfate liposome of modifying 22.0 ± 1.7
R
10H
3The vincristine sulfate liposome of modifying 23.1 ± 1.2
R
11H
3The vincristine sulfate liposome of modifying 24.2 ± 2.6
R
12H
3The vincristine sulfate liposome of modifying 25.1 ± 2.4
Embodiment 6
Wear film peptide R with hyaluronate sodium, the tumor cell selectivity that reaches of the present invention
8H
3The complex liposome constructed with Paclitaxel liposome is that example illustrates its tumor-targeting in the intravital tissue distribution of tumor-bearing mice.
Add 1.39gRinkamide-MBHA resin (0.74mmol/g) in 50mL side band arm, arm have the round-bottomed flask of sand plate filter element, added the 20mLDMF swelling 10 minutes, sucking filtration removes solvent.Add 20mL 20% piperidines/DMF solution then, stirred sucking filtration 30 minutes.With DMF washing resin 6 times, sucking filtration removes solvent.(Pbf is 2 to add 2.00g (3.09mmol) Fmoc-Arg (Pbf)-OH in reaction bulb; 2; 4; 6; 7-pentamethyl Dihydrobenzofuranes-5-sulfonyl), 0.64g (3.09mmol) dicyclohexylcarbodiimide and 0.42g (3.09mmol) HOBt (I-hydroxybenzotriazole) and 20mLDMF, stir under the room temperature and carry out condensation reaction.React the resin that takes a morsel after 2 hours and carry out the 1,2,3-indantrione monohydrate chromogenic reaction, the result shows that condensation reaction is complete.Sucking filtration is used DMF washing resin 6 times, and sucking filtration removes solvent.(protection) aminoacid of C end is connected on the resin.Reactions steps repeats top-operation to second of being connected with then that C held in back to last (protection) aminoacid, promptly from 20% piperidines/DMF solution deprotection, after the 1,2,3-indantrione monohydrate colour test with the DMF washing only.If it is incomplete that the 1,2,3-indantrione monohydrate colour test shows condensation, then can prolong the condensation time until fully.All with the Fmoc protection, aminoacid is followed successively by all amino acid whose a-amino: Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-His (Trt)-OH, Fmoc-His (Trt)-OH, Fmoc-His (Trt)-OH.Take off the Fmoc protection at last, drop into stearic acid and N end amino and finish condensation reaction.Polypeptide resin is washed for the third time with methanol, vacuum drying.
The above-mentioned resin that has connect peptide is joined in the 50mL round-bottomed flask, add 15mL reagent K (TFA/ thioanisole/EDT/ phenol/water=87.5/5.5/2.5/2.5/2.5 (v/v)), stirred 3 hours under the room temperature.Filter, collect filtrate.With give a baby a bath on the third day after its birth time resin of trifluoroacetic acid.Merging filtrate revolves steaming with filtrate and removes most trifluoroacetic acid, and adding volume is that 8-10 ether sedimentation doubly is connected to stearic polypeptide, puts into refrigerator overnight.Centrifugal, remove ether, vacuum drying obtains the crude product that N holds the RRRRRRRRHHH of the modification of being modified by stearic acid.
The above-mentioned product of 100mg is dissolved in the 2mL pure water, uses preparation reversed-phase HPLC purification, collect the reservation main peak, collect liquid and contract through revolving inspissation, lyophilization gets the pure product that N holds the RRRRRRRRHHH that is modified by stearic acid then.The mass spectrum of pure product shows that its molecular weight is 2143, conforms to value of calculation.The final N of acquisition holds the tumor cell selectivity of being modified by stearic acid to wear film peptide R
8H
3
Take by weighing injection soybean phospholipid, cholesterol and paclitaxel in eggplant type bottle according to recipe quantity, add a certain amount of chloroform it is fully dissolved, and the adding bead is some.Vacuum rotary steam 1 hour is waved except that organic solvent formation medicine membrane of lipoprotein.Finish back continuation evacuation and spend the night, to remove trace organic solvents.The hand hydration of 10mL pure water room temperature, till cleaning to eggplant type bottle wall, sample is uniform emulsion.Probe is ultrasonic to reduce particle diameter in the ice-water bath.3000rpm is centrifugal to remove the ultrasonic metal fillings that may drop of probe then.Get supernatant and cross 0.45 μ m respectively, each once can make Paclitaxel liposome 0.22 μ m polycarbonate leaching film.
Precision takes by weighing 8mg N and holds the tumor cell selectivity of being modified by stearic acid to wear film peptide R
8H
3, and it fully is dissolved in the distilled water of 2mL.Under room temperature, this solution slowly is added drop-wise in the Paclitaxel liposome dispersion that makes, the dropping process keeps magnetic agitation constantly.Dropping finishes the back and continues to stir 30min, this dispersion is hatched at 4 ℃ can make the Paclitaxel liposome that the tumor cell selectivity is worn the modification of film peptide in 8 hours then.The envelop rate of liposome before and after modifying and the investigation result of stability are shown that the tumor cell selectivity is worn film peptide R
8H
3Modification to Paclitaxel liposome does not produce appreciable impact to the physicochemical property of common Paclitaxel liposome, and envelop rate is greater than 96%, and room temperature is placed 24 hours percolation ratios less than 3%.
It is 10 that precision takes by weighing relative molecular mass
4Hyaluronate sodium 10mg, be dissolved in the distilled water of 4mL, regulate pH value to 6.0.Under room temperature, this solution slowly is added drop-wise to the tumor cell selectivity that makes and wears film peptide R
8H
3In the dispersion of the Paclitaxel liposome of modifying, the dropping process keeps magnetic agitation constantly.Continuation stirring 30min promptly was able to hyaluronate sodium after dropping finished, the tumor cell selectivity is worn film peptide R
8H
3The complex liposome constructed with Paclitaxel liposome.This complex liposome mean diameter is 168.4nm, and the zeta current potential is-18.7mV, and polydispersity index PDI is 0.288, and the paclitaxel envelop rate is 96.5%.Sucrose with 1% is freeze drying protectant, above-mentioned complex liposome is prepared into lyophilized powder preserves, and the glucose solution with 5% during use redissolves for the injection use.
With commercially available Paclitaxel liposome is contrast, and the paclitaxel complex liposome of method for preparing is investigated in the intravital tissue distribution of tumor-bearing mice.Choosing the oxter inoculation has 90 of the tumor-bearing mices (n=3) of S180 sarcoma, fasting 12 hours.Press 20mg/kg dosage respectively commercially available Paclitaxel liposome of tail vein injection and paclitaxel complex liposome, after the administration respectively at 5,15,30min, 1,2,4,8,12,24, after eye socket was got blood in 48 hours, disconnected neck was put to death, and gets the heart, liver, spleen, lung, kidney, the tumor sample of tumor-bearing mice, normal saline flushing, filter paper blot its moisture.Precision is weighed respectively, adds normal saline 1.5mL and makes homogenate.Precision is measured 0.8mL (liver is got 0.5mL) homogenate, and blood plasma is got 150 μ L, adds the interior mark of the diazepam liquid 50 μ L of variable concentrations respectively, behind the mixing, add t-butyl methyl ether 4mL, vortex 5min, the centrifugal 10min of 3000r/min, get organic facies 3mL, volatilize in 40 ℃ of decompressions, residue dissolves with mobile phase 200 μ L, vortex 1min, the centrifugal 10min of 10000r/min gets supernatant 20 μ L sample introductions, analyzes by chromatographic condition in the body.
The targeting evaluation of drug-supplying system adopts targeting efficient (Te) as evaluating.The pharmaceutical preparation of Te value representation is to the selectivity of target organ and non-target organ.The Te value is big more, and selectivity is strong more.The ratio of the AUC of each organ of this experiment exam and plasma A UC, i.e. Te=AUC
Organ/ AUC
Blood plasmaIts result as shown in Table 2.
The evaluation of table two targeting
By the result of table two as can be known, the paclitaxel complex liposome apparently higher than commercially available Paclitaxel liposome, and is starkly lower than commercially available Paclitaxel liposome to the targeting efficient Te of heart, kidney and other organs to the targeting efficient Te of tumor.This shows that the paclitaxel complex liposome has higher targeting to tumor, and reduced toxicity heart, kidney and other organs.
Embodiment 7
Wear film peptide R with hyaluronate sodium, the tumor cell selectivity that reaches of the present invention
6H
3The complex liposome constructed with Paclitaxel liposome is that example illustrates its tumor-targeting in the intravital tissue distribution of tumor-bearing mice.
Add 1.13gRinkamide-MBHA resin (0.74mmol/g) in 50mL side band arm, arm have the round-bottomed flask of sand plate filter element, added the 20mLDMF swelling 10 minutes, sucking filtration removes solvent.Add 20mL 20% piperidines/DMF solution then, stirred sucking filtration 30 minutes.With DMF washing resin 6 times, sucking filtration removes solvent.(Pbf is 2 to add 1.63g (2.51mmol) Fmoc-Arg (Pbf)-OH in reaction bulb; 2; 4; 6; 7-pentamethyl Dihydrobenzofuranes-5-sulfonyl), 0.52g (2.51mmol) dicyclohexylcarbodiimide and 0.34g (2.51mmol) HOBt (I-hydroxybenzotriazole) and 20mLDMF, stir under the room temperature and carry out condensation reaction.React the resin that takes a morsel after 2 hours and carry out the 1,2,3-indantrione monohydrate chromogenic reaction, the result shows that condensation reaction is complete.Sucking filtration is used DMF washing resin 6 times, and sucking filtration removes solvent.(protection) aminoacid of C end is connected on the resin.Reactions steps repeats top-operation to second of being connected with then that C held in back to last (protection) aminoacid, promptly from 20% piperidines/DMF solution deprotection, after the 1,2,3-indantrione monohydrate colour test with the DMF washing only.If it is incomplete that the 1,2,3-indantrione monohydrate colour test shows condensation, then can prolong the condensation time until fully.All with the Fmoc protection, aminoacid is followed successively by all amino acid whose a-amino: Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-His (Trt)-OH, Fmoc-His (Trt)-OH, Fmoc-His (Trt)-OH.Take off the Fmoc protection at last, drop into stearic acid and N end amino and finish condensation reaction.Polypeptide resin is washed for the third time with methanol, vacuum drying.
The above-mentioned resin that has connect peptide is joined in the 50mL round-bottomed flask, add 15mL reagent K (TFA/ thioanisole/EDT/ phenol/water=87.5/5.5/2.5/2.5/2.5 (v/v)), stirred 3 hours under the room temperature.Filter, collect filtrate.With give a baby a bath on the third day after its birth time resin of trifluoroacetic acid.Merging filtrate revolves steaming with filtrate and removes most trifluoroacetic acid, and adding volume is that 8-10 ether sedimentation doubly is connected to stearic polypeptide, puts into refrigerator overnight.Centrifugal, remove ether, vacuum drying obtains the crude product that N holds the RRRRRRHHH that is modified by stearic acid.
The above-mentioned product of 100mg is dissolved in the 2mL pure water, uses preparation reversed-phase HPLC purification, collect the reservation main peak, collect liquid and contract through revolving inspissation, lyophilization gets the pure product that N holds the RRRRRRHHH that is modified by stearic acid then.The mass spectrum of pure product shows that its molecular weight is 1794, conforms to value of calculation.The final N of acquisition holds the tumor cell selectivity of being modified by stearic acid to wear film peptide R
6H
3
Take by weighing injection soybean phospholipid, cholesterol and paclitaxel in eggplant type bottle according to recipe quantity, add a certain amount of chloroform it is fully dissolved, and the adding bead is some.Vacuum rotary steam 1 hour is waved except that organic solvent formation medicine membrane of lipoprotein.Finish back continuation evacuation and spend the night, to remove trace organic solvents.The hand hydration of 10mL pure water room temperature, till cleaning to eggplant type bottle wall, sample is uniform emulsion.Probe is ultrasonic to reduce particle diameter in the ice-water bath.3000rpm is centrifugal to remove the ultrasonic metal fillings that may drop of probe then.Get supernatant and cross 0.45 μ m respectively, each once can make the liposome paclitaxel 0.22 μ m polycarbonate leaching film.
Precision takes by weighing 10mg N and holds the tumor cell selectivity of being modified by stearic acid to wear film peptide R
6H
3, and it fully is dissolved in the distilled water of 2mL.Under room temperature, this solution slowly is added drop-wise in the Paclitaxel liposome dispersion that makes, the dropping process keeps magnetic agitation constantly.Dropping finishes the back and continues to stir 30min, this dispersion is hatched at 4 ℃ can make the Paclitaxel liposome that the tumor cell selectivity is worn the modification of film peptide in 8 hours then.The envelop rate of liposome before and after modifying and the investigation result of stability are shown that the tumor cell selectivity is worn film peptide R
6H
3Modification to Paclitaxel liposome does not produce appreciable impact to the physicochemical property of common Paclitaxel liposome, and envelop rate is greater than 96%, and room temperature is placed 24 hours percolation ratios less than 3%.
It is 10 that precision takes by weighing relative molecular mass
4Hyaluronate sodium 10mg, be dissolved in the distilled water of 4mL, regulate pH value to 6.0.Under room temperature, this solution slowly is added drop-wise to the tumor cell selectivity that makes and wears film peptide R
6H
3In the dispersion of the Paclitaxel liposome of modifying, the dropping process keeps magnetic agitation constantly.Continuation stirring 30min promptly was able to hyaluronate sodium after dropping finished, the tumor cell selectivity is worn film peptide R
6H
3The complex liposome constructed with Paclitaxel liposome.This complex liposome mean diameter is 160.7nm, and the zeta current potential is-21.3mV, and polydispersity index PDI is 0.278, and the paclitaxel envelop rate is 97.0%.Sucrose with 1% is freeze drying protectant, above-mentioned complex liposome is prepared into lyophilized powder preserves, and the glucose solution with 5% during use redissolves for the injection use.
With commercially available Paclitaxel liposome is contrast, and the paclitaxel complex liposome of method for preparing is investigated in the intravital tissue distribution of tumor-bearing mice.Choosing the oxter inoculation has 90 of the tumor-bearing mices (n=3) of H22 hepatocarcinoma, fasting 12 hours.Press 20mg/kg dosage respectively commercially available Paclitaxel liposome of tail vein injection and paclitaxel complex liposome, after the administration respectively at 5,15,30min, 1,2,4,8,12,24, after eye socket was got blood in 48 hours, disconnected neck was put to death, and gets the heart, liver, spleen, lung, kidney, the tumor sample of tumor-bearing mice, normal saline flushing, filter paper blot its moisture.Precision is weighed respectively, adds normal saline 1.5mL and makes homogenate.Precision is measured 0.8mL (liver is got 0.5mL) homogenate, and blood plasma is got 150 μ L, adds the interior mark of the diazepam liquid 50 μ L of variable concentrations respectively, behind the mixing, add t-butyl methyl ether 4mL, vortex 5min, the centrifugal 10min of 3000r/min, get organic facies 3mL, volatilize in 40 ℃ of decompressions, residue dissolves with mobile phase 200 μ L, vortex 1min, the centrifugal 10min of 10000r/min gets supernatant 20 μ L sample introductions, analyzes by chromatographic condition in the body.
The targeting evaluation of drug-supplying system adopts targeting efficient (Te) as evaluating.The pharmaceutical preparation of Te value representation is to the selectivity of target organ and non-target organ.The Te value is big more, and selectivity is strong more.The ratio of the AUC of each organ of this experiment exam and plasma A UC, i.e. Te=AUC
Organ/ AUC
Blood plasmaIts result as shown in Table 3.
The evaluation of table three targeting
By the result of table three as can be known, the paclitaxel complex liposome apparently higher than commercially available Paclitaxel liposome, and is starkly lower than commercially available Paclitaxel liposome to the targeting efficient Te of heart, kidney and other organs to the targeting efficient Te of tumor.This shows that the paclitaxel complex liposome has higher targeting to tumor, and reduced toxicity heart, kidney and other organs.
Embodiment 8
Wear film peptide (R with hyaluronate sodium, tumor cell selectivity
5H
3, R
6H
3, R
7H
3, R
8H
3, R
9H
3, R
10H
3, R
11H
3, R
12H
3, R is at N end or C end) with the constructed complex liposome of hydroxy camptothecin liposome be the antitumor efficacy that example illustrates this preparation at the intravital tumor killing effect of tumor-bearing mice.
Take by weighing injection lecithin, cholesterol and hydroxy camptothecin in eggplant type bottle according to recipe quantity, add a certain amount of ethanol it is fully dissolved, and the adding bead is some.Vacuum rotary steam 1 hour is waved except that organic solvent formation medicine membrane of lipoprotein.Finish back continuation evacuation and spend the night, to remove trace organic solvents.The hand hydration of 10mL phosphate buffered solution (pH4.5) room temperature, till cleaning to eggplant type bottle wall, sample is uniform emulsion.Adopt experiment type high pressure homogenizer to reduce particle diameter.3000rpm is centrifugal to remove the ultrasonic metal fillings that may drop of probe then.Get supernatant and cross 0.45 μ m respectively, each once can make the hydroxy camptothecin liposome 0.22 μ m polycarbonate leaching film.
Precision takes by weighing a certain amount of N and holds the tumor cell selectivity of being modified by stearic acid to wear film peptide R
5H
3(R is at C end or N end), and it fully is dissolved in the distilled water of 2mL.Under room temperature, this solution slowly is added drop-wise in the hydroxy camptothecin liposome dispersion that makes, the dropping process keeps magnetic agitation constantly.Drip to finish the back and continue to stir 30min, then this dispersion is hatched at 4 ℃ and can make the tumor cell selectivity in 8 hours and wear film peptide R
5H
3The hydroxy camptothecin liposome that (R is at C end or N end) modified.The envelop rate of liposome before and after modifying and the investigation result of stability are shown that the tumor cell selectivity is worn film peptide R
5H
3(R is at C end or N end) do not produce appreciable impact to the physicochemical property of common hydroxy camptothecin liposome to the modification of hydroxy camptothecin liposome, and envelop rate is greater than 90%, and room temperature is placed 24 hours percolation ratios less than 3%.
It is 10 that precision takes by weighing relative molecular mass
4Hyaluronate sodium 10mg, be dissolved in the distilled water of 4mL, regulate pH value to 6.0.Under room temperature, this solution slowly is added drop-wise to the tumor cell selectivity that makes and wears film peptide R
5H
3In the dispersion of the hydroxy camptothecin liposome that (R is at C end or N end) modified, the dropping process keeps magnetic agitation constantly.Continuation stirring 30min promptly was able to hyaluronate sodium after dropping finished, the tumor cell selectivity is worn film peptide R
5H
3(R is at C end or N end) and the constructed complex liposome of hydroxy camptothecin liposome.This complex liposome mean diameter is 187.5nm, and the zeta current potential is-20.4mV, and polydispersity index PDI is 0.259, and the paclitaxel envelop rate is 92.5%.Sucrose with 1% is freeze drying protectant, above-mentioned complex liposome is prepared into lyophilized powder preserves, and the glucose solution with 5% during use redissolves for the injection use.
Hyaluronate sodium, other tumor cell selectivitys are worn film peptide (R
6H
3, R
7H
3, R
8H
3, R
9H
3, R
10H
3, R
11H
3, R
12H
3, R is at N end or C end) and can adopt identical method to prepare with the constructed complex liposome of hydroxy camptothecin liposome.
In addition, adopt identical method to prepare the hydroxy camptothecin liposome, and worn film peptide R with N end by what stearic acid was modified as stated above
8Prepare R together with hyaluronate sodium
8The hydroxy camptothecin liposome of modifying.
(EAC) is model with the mice ehrlich ascites tumor: take out well-grown ehrlich ascites tumor cell inoculation in female NIH strain intraperitoneal mouse (every milliliter about 5 * 10
6Individual, every 0.2mL), get ascites after 8 days, with the normal saline dilution, be re-seeded into another healthy mice intraperitoneal, passed for 3 generations altogether.60 female NIH strain white mice are weighed, be divided into 12 groups at random, 5 every group; From the mouse peritoneal that goes down to posterity, extract ascites tumor out and count, be diluted to every milliliter 4 * 10
6Individual, be inoculated in right side of mice back subcutaneous (every 0.2mL); With various hydroxy camptothecin complex liposomes, the R that makes
8Hydroxy camptothecin liposome, common hydroxy camptothecin liposome and the alkyl camptothecine injection modified are pressed the 5mg/kg hydroxy camptothecin, and in the 7th day single-dose in inoculation back, the blank group is injected isopyknic normal saline (blank).After inoculating for two weeks the disconnected neck of mice is put to death, local 75% ethanol disinfection of using, get the subcutaneous tumors piece with shears, peel off fibrous tissue and weigh, tumor control rate is pressed average weight * 100% of suppression ratio %=(average weight of the average weight of blank group tumor-administration group tumor)/blank group tumor, and the tumor anharmonic ratio more as shown in Table 4.The result shows, gives the treatment of hydroxy camptothecin complex liposome 5mg/kg dosage pool on the 7th day behind the tumor cell inoculation, and tumor control rate is significantly higher than R
8Hydroxy camptothecin liposome, the conventional liposome of hydroxy camptothecin and the suppression ratio (P＜0.05) of injection dosage form administration group modified.Hence one can see that, wears the constructed complex liposome of film peptide and hydroxy camptothecin liposome with hyaluronate sodium, tumor cell selectivity and obviously be better than R at the intravital tumor killing effect of tumor-bearing mice
8The hydroxy camptothecin liposome of modifying, the conventional liposome and the injection dosage form of hydroxy camptothecin.
The tumor body of each administration group of table four is weighed (g) and tumor control rate (%), n=5,
Embodiment 9
Wear film peptide (R with hyaluronate sodium, tumor cell selectivity
5H
3, R
6H
3, R
7H
3, R
8H
3, R
9H
3, R
10H
3, R
11H
3, R
12H
3, R is at N end or C end) with the constructed complex liposome of Paclitaxel liposome be the antitumor efficacy that example illustrates this preparation at the intravital tumor killing effect of tumor-bearing mice.
Take by weighing injection soybean phospholipid, cholesterol and paclitaxel in eggplant type bottle according to recipe quantity, add a certain amount of chloroform it is fully dissolved, and the adding bead is some.Vacuum rotary steam 1 hour is waved except that organic solvent formation medicine membrane of lipoprotein.Finish back continuation evacuation and spend the night, to remove trace organic solvents.The hand hydration of 10mL pure water room temperature, till cleaning to eggplant type bottle wall, sample is uniform emulsion.Probe is ultrasonic to reduce particle diameter in the ice-water bath.3000rpm is centrifugal to remove the ultrasonic metal fillings that may drop of probe then.Get supernatant and cross 0.45 μ m respectively, each once can make the liposome paclitaxel 0.22 μ m polycarbonate leaching film.
Precision takes by weighing 10mg N end and is worn film peptide R by the tumor cell selectivity of just fat acid modification
5H
3(R is at C end or N end), and it fully is dissolved in the distilled water of 2mL.Under room temperature, this solution slowly is added drop-wise in the Paclitaxel liposome dispersion that makes, the dropping process keeps magnetic agitation constantly.Dropping finishes the back and continues to stir 30min, this dispersion is hatched at 4 ℃ can make the Paclitaxel liposome that the tumor cell selectivity is worn the modification of film peptide in 8 hours then.The envelop rate of liposome before and after modifying and the investigation result of stability are shown that the tumor cell selectivity is worn film peptide R
5H
3Modification to Paclitaxel liposome does not produce appreciable impact to the physicochemical property of common Paclitaxel liposome, and envelop rate is greater than 96%, and room temperature is placed 24 hours percolation ratios less than 3%.
It is 10 that precision takes by weighing relative molecular mass
4Hyaluronate sodium 10mg, be dissolved in the distilled water of 4mL, regulate pH value to 6.0.Under room temperature, this solution slowly is added drop-wise to the tumor cell selectivity that makes and wears film peptide R
5H
3In the dispersion of the Paclitaxel liposome of modifying, the dropping process keeps magnetic agitation constantly.Continuation stirring 30min promptly was able to hyaluronate sodium after dropping finished, the tumor cell selectivity is worn film peptide R
5H
3The complex liposome constructed with Paclitaxel liposome.This complex liposome mean diameter is 162.4nm, and the zeta current potential is-19.3mV, and polydispersity index PDI is 0.265, and the paclitaxel envelop rate is 98.4%.Sucrose with 1% is freeze drying protectant, above-mentioned complex liposome is prepared into lyophilized powder preserves, and the glucose solution with 5% during use redissolves for the injection use.
Hyaluronate sodium, other tumor cell selectivitys are worn film peptide (R
6H
3, R
7H
3, R
8H
3, R
9H
3, R
10H
3, R
11H
3, R
12H
3, R is at N end or C end) and can adopt identical method to prepare with the constructed complex liposome of Paclitaxel liposome.
In addition, adopt identical method to prepare Paclitaxel liposome, and worn film peptide R with N end by what stearic acid was modified as stated above
8Prepare R together with hyaluronate sodium
8The Paclitaxel liposome of modifying.
It is subcutaneous in BABL/C nude mice back to get well-grown human oophoroma cell line COC1 cell inoculation.After 5 weeks, the inoculation position tumor begins growth, and diameter of tumor reaches about 1.5cm during the 8th week, can be used for experiment.Totally 60 of the nude mices of said method success inoculated tumour are divided into 12 groups, 5 every group at random.
With various paclitaxel complex liposomes, the R that makes
8Paclitaxel liposome, common Paclitaxel liposome and the paclitaxel injection modified are pressed the 10mg/kg paclitaxel, the administration of single tail vein injection, and the blank group is injected isopyknic normal saline (blank).After one week of administration the disconnected neck of mice is put to death, local 75% ethanol disinfection of using, get the subcutaneous tumors piece with shears, peel off fibrous tissue and weigh, tumor control rate is pressed average weight * 100% of suppression ratio %=(average weight of the average weight of blank group tumor-administration group tumor)/blank group tumor, and the tumor anharmonic ratio more as shown in Table 5.The result shows that give paclitaxel complex liposome 10mg/kg dosage treatment behind the tumor cell inoculation, tumor control rate is significantly higher than R
8The suppression ratio (P＜0.05) of Paclitaxel liposome, paclitaxel conventional liposome and the paclitaxel injection dosage form administration group of modifying.Hence one can see that, wears the constructed complex liposome of film peptide and Paclitaxel liposome with hyaluronate sodium, tumor cell selectivity and obviously be better than R at the intravital tumor killing effect of tumor-bearing mice
8Paclitaxel liposome, paclitaxel conventional liposome and the paclitaxel injection dosage form of modifying.
The tumor body of each administration group of table five is weighed (g) and tumor control rate (%), n=5,
Claims (10)
1. complex liposome that contains antitumor drug, it is characterized in that: it contains antitumor drug, lipid, hyaluronic acid or hyaluronate sodium and the tumor cell selectivity is worn the film peptide.
2. complex liposome according to claim 1, it is characterized in that: press mass ratio, it contains the antitumor drug of 0.01%-30%, the lipid of 1%-98%, the 0.001%-20% hyaluronate sodium, the tumor cell selectivity of 0.001%-20% is worn the film peptide, the alcohol of 0-20%, the antioxidant of 0-20%, the antiseptic of 0-20%, the pH regulator agent of 0-20%, the phase transformation regulator of 0-20%, the long recycled material of 0-20%, the isoosmotic adjusting agent of 0-20%, the lyophilizing proppant of 0-95%, the water of 0-95%.
3. complex liposome according to claim 1 and 2 is characterized in that described antitumor drug is selected from one or more in the following medicine: paclitaxel, Docetaxel, actinomycin D, doxorubicin, epirubicin, daunorubicin, vincristine, vinorelbine, cisplatin, oxaliplatin, methotrexate, fluorouracil, mercaptopurine, cytosine arabinoside, doxifluridine, etoposide, teniposide, camptothecine, hydroxy camptothecin, topotecan, irinotecan, mitoxantrone, cyclophosphamide, ifosfamide, ametycin, busulfan, lomustine, carmustine, semustine, nimustine, Ranimustine, gemcitabine, capecitabine, decitabine, ancitabine, cisplatin, bleomycin, Bleomycin A5, gamlogic acid, the various pharmaceutical salts of neogambogic acid and these medicines.
4. complex liposome according to claim 1 and 2 is characterized in that described lipid is phospholipid and cholesterol, and phospholipid is selected from one or more in natural phospholipid, semi-synthetic phospholipid and the complete synthesis phospholipid.
5. complex liposome according to claim 1 and 2, the relative molecular mass that it is characterized in that described hyaluronic acid or hyaluronate sodium is from 4 * 10
3Dalton to 2 * 10
7Dalton.
6. complex liposome according to claim 1 and 2, it is characterized in that described tumor cell selectivity wears the film peptide and for tumor cell or tissue stronger cell membrane penetration is arranged, maybe can not see through the cell membrane penetration of normal cell or tissue is more weak.
7. complex liposome according to claim 1 and 2 is characterized in that described tumor cell selectivity wears the film peptide and be selected from the following polypeptide one or more: RRRRRHHH (R
5H
3, R is at C end or N end), RRRRRRHHH (R
6H
3, R is at C end or N end), RRRRRRRHHH (R
7H
3, R is at C end or N end), RRRRRRRRHHH (R
8H
3, R is at C end or N end), RRRRRRRRRHHH (R
9H
3, R is at C end or N end), RRRRRRRRRRHHH (R
10H
3, R is at C end or N end), RRRRRRRRRRRHHH (R
11H
3, R is at C end or N end), RRRRRRRRRRRRHHH (R
12H
3, R is at C end or N end), their N end is by fatty acid modifying.
8. fatty acid according to claim 7 is characterized in that: the carbochain of fatty acid can contain 8-18 carbon atom, is preferably stearic acid.
9. complex liposome according to claim 1 and 2, its preparation method comprise the method that organic solvent injection method, film dispersion method, reverse evaporation, active loading method, multigelation method, lyophilization and above-mentioned each method combine.
10. claim 1 or 2 described complex liposomes is characterized in that preparing the pharmaceutical preparation for the treatment of tumor.
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