CN104587446A - Temperature-sensitive lipopeptid, lipidosome containing lipopeptid and application of temperature-sensitive lipopeptid and lipidosome - Google Patents
Temperature-sensitive lipopeptid, lipidosome containing lipopeptid and application of temperature-sensitive lipopeptid and lipidosome Download PDFInfo
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Abstract
The invention provides a temperature-sensitive lipopeptid, wherein a molecular structure of the lipopeptid is CH3-(CH2)n-(VXXLXXX)m; n is 4-10; m is 3-5; V is valine; L is leucine; and X is hydrophilic amino acid except for the valine and the leucine. The invention further provides a temperature-sensitive lipopeptid-lipidosome formed by self-assembly of lipopeptid and phospholipid at the molar ratio of (1-50) to 200. Through selecting different phospholipids, regulating and controlling the phospholipid ratio, adopting different lipopeptids and regulating and controlling the ratio of lipopeptids to phospholipids, the lipopeptid-lipidosome with different phase transition temperatures can be obtained, so that the target of targeted drug delivery is reached.
Description
Technical field
The invention belongs to physical chemistry field, be specifically related to the temperature sensitivity lipopeptid of synthesis and the application in liposome thereof.
Background technology
Liposome is a kind of new medicinal preparation, is the vesicle packaging medicine molecule utilizing phospholipid bilayer film to be formed.Basic structure due to organism plasma membrane is also phospholipid bilayer film, and liposome has the structure similar with biological cell, therefore has good biocompatibility.
In the various novel lipides of research, temperature sensitive liposome is a rising branch, and it effectively make use of the double dominant of liposome and thermotherapy to improve therapeutic effect, reduces toxic and side effects.Common temperature sensitivity liposome is generally made up of phospholipid and cholesterol, each phospholipid all has specific phase transition temperature, phospholipid is utilized to have certain phase transition temperature, different phospholipid and cholesterol are mixed according to different proportionings, the phase transition temperature of the liposome obtained is also different.As Gerben A.Koning
[1], David Needham
[2]deng the difference once mentioned in the literature on the phase transition temperature utilizing various phospholipid, proportion of composing by phospholipid regulates the phase transition temperature Tm of liposome, although there is the effect of certain increase burst size at its more than Tm, but when lower than Tm temperature, the leakage rate of its medicine also can correspondingly improve, drug side effect is strengthened, so, there is document also to report and synthesized Thermo-sensitive block copolymer
[3], be inserted in liposome bilayer membrane, the temperature control release effect of such liposome increases really, but has the vivo degradation problem of high molecular polymer.
The present invention is based on the pluses and minuses of above liposome, design and synthesis has the amphiphilic molecules of Thermo-sensitive polypeptide, and it is formed Coliposomes with phospholipid mixing self assembly, this Thermo-sensitive liposome is in order to pharmaceutical carrier, under extraneous high temperature stimulation, complete the object of release internal substance rapidly.
Summary of the invention
The object of the present invention is to provide a kind of lipopeptid with temperature sensitivity.
A further object of the present invention is to provide a kind of lipopeptid-liposome with temperature sensitivity.
Further object of the present invention is to provide described temperature sensitivity lipopeptid-liposome preparing the purposes on targeted drug.
Present inventor devises a kind of lipopeptid with temperature sensitivity, and its molecular structure is CH
3-(CH
2) n-(VXXLXXX) m, wherein n:4 ~ 10, m:3 ~ 5, V: valine; L: leucine, X: except the hydrophilic amino acid except valine and leucine.
This lipopeptid with temperature sensitivity of present inventor's design entrusts the synthesis of Ai Te bio tech ltd, Nanjing.
This lipopeptid with temperature sensitivity provided by the invention is made up of carbochain and polypeptide, and have amphipathic, one end is hydrophilic peptide chain, and the other end is hydrophobic carbon chain.
The present invention also provides a kind of lipopeptid-liposome with temperature sensitivity, is CH by molecular structure
3-(CH
2) n-(VXXLXXX) m (wherein n:4 ~ 10, m:3 ~ 5, V: valine; L: leucine, X: the hydrophilic amino acid except except valine and leucine) temperature sensitivity lipopeptid and phospholipid formed with the mol ratio self assembly of 1 ~ 50:200.
In temperature sensitivity lipopeptid-liposome of the present invention, phospholipid is selected from HSPC, DOPC, DPPE, DPPC and DSPG.
Described lipopeptid-liposome is embedded in liposome bilayer membrane by lipopeptid and is formed.
The invention provides the preparation method of this lipopeptid-liposome.
Lipopeptid-liposome of the present invention can be the liposome of not bag medicine, and it obtains by the following method:
1) lipopeptid and phospholipid are dissolved in the organic solvent being selected from chloroform, absolute methanol and ethanol respectively, in 30-60 DEG C of outstanding steaming 30-60min after gained solution fully mixes, obtain lipopeptid-liposome membrane;
2) solvent evaporate off in vacuo, continue to drain after lipopeptid-liposome deposition bottle wall, with tris-HCl solubilize lipopeptid-liposome membrane, 30-60 DEG C of ultrasonic disperse 10-30min, obtains lipopeptid-liposome solutions;
3) with filter by step 2) lipopeptid-liposome solutions of gained filters by the extruder of filter membrane diameter 200nm.
Preferably, also add the cholesterol being dissolved in organic solvent when described lipopeptid and phospholipid mixing and be mixed together, more preferably adopt catatonic cholesterol.
Select catatonic cholesterol to make liposome positively charged, the liposome made like this is more stable than uncharged liposome.
Lipopeptid-liposome solutions provided by the invention is after the extruder of filter membrane diameter 200nm is filtered, and the size controlling of liposome is at about 200nm.
In a preferred embodiment, comprise the steps:
1) in a round-bottomed flask, phospholipid and the catatonic cholesterol of mol ratio 4:1 is added, with organic solvent dissolution, add the lipopeptid of other organic solvent dissolution, the ratio of lipopeptid and phospholipid is 1 ~ 50:200, fully mixes, 30-60 DEG C revolve steam more than 45min stand-by;
2) by the lipopeptid of step 1 gained-liposome membrane evacuation, treat that solvent volatilizees completely, continue to drain after lipopeptid-liposome deposition bottle wall, then use tris solubilize, 30-60 DEG C of ultrasonic disperse 10-30min, obtains lipopeptid-liposome solutions;
3) with filter by step 2) lipopeptid-liposome solutions of gained extruder (filter membrane diameter 200nm) filters, and controls the particle diameter of liposome near 200nm.
The liposome of what lipopeptid-liposome provided by the invention was more practical is bag medicine, it disperses aquation legal system to obtain by thin film, comprises the steps:
1) lipopeptid and phospholipid are dissolved in the organic solvent being selected from chloroform, absolute methanol and ethanol respectively, in 30-60 DEG C of outstanding steaming 30-60min after gained solution fully mixes, obtain lipopeptid-liposome membrane;
2) solvent evaporate off in vacuo, continues to drain after lipopeptid-liposome deposition bottle wall, adds 5ml (NH
4)
2sO
4solution aquation lipopeptid-liposome membrane, 30-60 DEG C of ultrasonic disperse 10-30min, obtains lipopeptid-liposome solutions;
3) with filter by step 2) lipopeptid-liposome solutions of gained filters by the extruder of filter membrane diameter 200nm.
Preferably, also add the cholesterol being dissolved in organic solvent when described lipopeptid and phospholipid mixing and be mixed together, more preferably adopt catatonic cholesterol.
Lipopeptid-liposome solutions provided by the invention is after the extruder of filter membrane diameter 200nm is filtered, and the size controlling of liposome is at about 200nm.
A specific embodiment comprises the steps:
1) in a round-bottomed flask, the phospholipid of additional proportion 4:1 and catatonic cholesterol, with organic solvent dissolution, add the lipopeptid of other organic solvent dissolution, the ratio of lipopeptid and phospholipid is xx, rotary evaporation removing organic solvent in 45 DEG C of waters bath with thermostatic control, continues to drain after liposome deposition bottle wall;
2), after taking off round-bottomed flask, put into 60 DEG C of ultrasonic preheatings of water-bath, in bottle, add 5ml (NH
4)
2sO
4solution aquation liposome, ultrasonic disperse 30min;
3) with filter, the extruder of lipopeptid-liposome solutions filter membrane diameter 200nm of gained is filtered, control the particle diameter of liposome near 200nm.
The medicine-carrying method of lipopeptid-liposome provided by the invention can be active or Passive loading method.
The drug-loaded liposome of packaging medicine provided by the invention obtains by the following method:
1) by the liposome of bag medicine after dialysis, be placed in 60 DEG C of water-baths, with medicine: the ratio of liposome=1:20 drips drug solution, hatches 30min in a water bath after being added dropwise to complete, every 10min concussion mixing once;
2) non-encapsulated medicine is separated by dextran microgel column.
A preferred embodiment of the present invention, makes medicament-carried nano liposome by thin-film ultrasonic aquation method, and in its preparation method, solvent evaporate off in vacuo is rotary evaporation in 45 DEG C of waters bath with thermostatic control, adds (NH
4)
2sO
4first ultrasonic preheating in 60 DEG C of water-baths before solution aquation, described ultrasonic disperse carries out 30min.
Brief description of drawings
Fig. 1 is circular dichroism spectra (CD) figure of pure lipopeptid at 6 DEG C ~ 92 DEG C.
Fig. 2 is circular dichroism spectra (CD) figure of lipopeptid-liposome at 6 DEG C ~ 92 DEG C.
Fig. 3 is transmission electron microscope (TEM) figure of liposome.
Fig. 4 is the drug release profiles of lipopeptid-liposome at 45 DEG C and 37 DEG C.
The present invention is based on the leucic characteristic of zipper, a kind of lipopeptid with temperature sensitivity of design and synthesis.Described lipopeptid is made up of carbochain and polypeptide, has amphipathic, and one end is hydrophilic peptide chain, and the other end is hydrophobic carbon chain.Every 7 aminoacid of the peptide end of the chain are one group, the 4th aminoacid is wherein leucine, polypeptide forms α-helixstructure in aqueous, every 7 aminoacid are 1 spiral, such leucine is just in time arranged in the side of spiral, because leucine is hydrophobic amino acid, a-spiral is combined between two by hydrophobic interaction.When 2 amino acid chains are arranged in parallel, just form dimer, along with temperature rises, a-helical structure is destroyed, and dimer is dismissed, and is referred to as the function with " slide fastener ".As seen from Figure 1, pure lipopeptid has characteristic peak at 192nm, 208nm and 222nm place, illustrates that lipopeptid has αhelix in tris solution.When the temperature increases, characteristic peak reduces, and helical structure is destroyed.By software processes, the phase transition temperature obtaining lipopeptid is 48.0 ± 0.1 DEG C.
Be inserted in liposome membrane by the lipopeptid with temperature sensitivity, the lipopeptid-liposome of formation has certain temperature sensitivity as pharmaceutical carrier, and when the transition temperature of temperature lower than lipopeptid-liposome, medicine does not leak; When the transition temperature of temperature higher than lipopeptid-liposome, because dimer is dismissed, medicine is released.As seen from Figure 2, when being inserted in liposome by lipopeptid, the characteristic peak of lipopeptid still exists, and illustrates that lipopeptid still remains αhelix in liposome membrane.By software processes, the phase transition temperature of lipopeptid-liposome is 45.0 ± 0.1 DEG C.
From the particle diameter through the visible lipopeptid-liposome of electron microscope picture of Fig. 3 greatly between 200 ~ 300nm.
Lipopeptid-liposome of the present invention is due to the interaction of lipopeptid and phospholipid, and when the transition temperature of temperature slightly lower than lipopeptid, lipopeptid just can be opened and form duct, and medicine therefrom discharges.Experimental result shows that such structural design makes the rate of release of medicine and burst size all than general temperature sensitivity lipid height.Each lipoid plastid 24 Hours drug burst size as seen from Figure 4, when ambient temperature is 37 DEG C, the release amount of medicine of liposome only 24.8%, also just 25.5% of lipopeptid-liposome; 45 DEG C time, the release amount of medicine of liposome slightly improves, and about 37.8%, lipopeptid-liposome reaches 70%.Result shows, after inserting lipopeptid, the drug stabilisation of holding of liposome increases, and when ambient temperature exceedes lipopeptid phase transition temperature, release amount increases greatly.
Mixed by the phospholipid of different phospholipid and different proportion, the liposome made also has different phase transition temperatures.
Due between different tissues (especially tumor cell and normal cell) because of metabolic rate different tissues temperature slightly difference, by selecting different phospholipid and regulation and control phospholipid ratio, and adopt the ratio of different lipopeptids and regulation and control lipopeptid and phospholipid, the liposome with different phase transition temperature can be obtained, thus reach the object of target administration.
Therefore, the present invention also provides lipopeptid-liposome preparing the purposes on targeted drug.
Temperature sensitivity liposome provided by the invention has excellent performance in drug release.
Detailed description of the invention
For a more detailed description to the present invention by embodiment below.These embodiments are only the descriptions to best mode for carrying out the invention, do not have any restriction to scope of the present invention.
1, term explanation
HSPC: hydrogenated soya phosphatide;
DSPC: distearoyl phosphatidylcholine (1,2-distearoyl-sn-glycero-3-phosphocholine);
DPPE: DPPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine);
DC-Chol: 3 β-[N-(dimethylamino ethane) carbamoyl cholesterol (Cholesteryl3 β-N-(di-methyl-amino-ethyl)-carbamate hydrochloride);
Dox: doxorubicin hydrochloride;
Tris: Tris
2, reagent explanation
HSPC, DSPC, DPPE buy from German lipoid company, purity >97%;
Tris, purity higher than 99.9%, lark prestige Science and Technology Ltd.;
Sephadex G-50, Beijing Rui Dahenghui development in science and technology company limited;
Leucine zipper lipopeptid, Ai Te bio tech ltd, Nanjing, purity is higher than 95%
DC-chol buys from Sigma company, purity >95%;
Chloroform, methanol, ethanol, analytical pure, Solution on Chemical Reagents in Shanghai company limited produces.
3, instrument explanation
Zetasizer Nano S type dynamic light scattering (DLS, Malvern company of Britain);
Circular dichroism spectrometer (CD, Applied Photophysics company of Britain);
Ultraviolet-uisible spectrophotometer (UV 1601PC, Japanese Shimadzu Corporation);
PH meter (PHS-3D, Shanghai Precision Scientific Apparatus Co., Ltd);
Vacuum desiccator (DZG-602, Shanghai is gloomy reliablely tests Instrument Ltd.);
Transmission electron microscope (JEM-1400, Japanese JEOL company);
Rotary Evaporators (N-1001, Shanghai Ai Lang Instrument Ltd.);
DSC differential scanning calorimeter (Q2000 differential scanning calorimeter, the U.S.)
High-frequency digitally controlled ultrasound wave (Kunshan Ultrasonic Instruments Co., Ltd.);
Magnetic stirring apparatus (Shanghai Si Le Instrument Ltd.).
Embodiment 1
Lipopeptid structure is [CH
3-(CH
2)
6-VAQLEVK-VAQLESK-VSKLESK-VSSLESK] the preparation of lipopeptid-liposome
the preparation of solution
1. the preparation of phospholipid solution: use analytical balance to take 9.04mg HSPC, 9.72mg DSPC, 0.85mgDPPE, be added in 5ml volumetric flask together, with chloroform: methanol is that the mixed solvent of 4:1 dissolves standardize solution.
2. the preparation of catatonic cholesterol solution: 3.3mg 3 β-[N-(dimethylamino ethane) carbamoyl cholesterol, with chloroform: methanol is that the mixed solvent dissolving of 4:1 is settled in 5ml volumetric flask to use analytical balance to take.
3. the preparation of lipopeptide solution: use analytical balance to take 0.39mg leucine zipper type lipopeptid [CH
3-(CH
2)
6-VAQLEVK-VAQLESK-VSKLESK-VSSLESK], be that dissolution with solvents is settled in 5ml volumetric flask with methanol.
4. the preparation of buffer solution: get 75.875mg tri-(methylol) aminomethane reagent, adds the dissolving of about 495ml deionized water magnetic agitation and obtains Tris solution; Getting 1ml concentration is that 36% ~ 38% concentrated hydrochloric acid solution ultra-pure water is diluted in 100ml volumetric flask; Be added drop-wise to by diluted hydrochloric acid solution in tris solution, dropping limit, limit measures the pH of solution, and the pH to mixed solution is 7.4, finally adds deionized water and is settled to 500ml, obtains the tris-HCl buffer solution that pH is 7.4, concentration is 1.25mM.
5. the preparation of Doxorubicin solution: get 25mg amycin (DOX) in 5ml volumetric flask, with the above-mentioned Tris-HCl buffer solution standardize solution prepared, obtains 5mg/ml Doxorubicin solution.Note lucifuge in process for preparation, prevent amycin from seeing photolysis.
6. the preparation of ammonium sulfate solution: take 0.528g (NH
4)
2sO
4in 20ml volumetric flask, add deionized water standardize solution, dissolve and be made into 0.2mol/l (NH
4)
2sO
4solution.
The preparation of 7.NaCl solution: take 22.5g NaCl, adds 2.5L deionized water, namely obtains 0.9mol/L NaCl solution after dissolving.
8. the preparation of dextran solution: take about 3g G50 glucosan, add deionized water with the ratio of 1:6, soak 6 hours, glucosan is expanded to 5 times of original volume, obtains dextran solution.
the preparation of liposome
This experiment adopts thin film aquation legal system for liposome.
1. the preparation of mixed solution: get 2.5ml phospholipid solution, 2.5ml catatonic cholesterol solution and 5ml lipopeptide solution (blank experiment does not need to get this solution) and add in 250ml round-bottomed flask, add 7.5ml chloroform again, stir, shake 5min, obtain mixed solution.
2. revolve steaming: being adjusted to 45 DEG C by revolving the bath temperature steaming instrument, stablizing 30min.Immersed by flask in 45 DEG C of waters bath with thermostatic control, rotary speed is set to 100r/min, is evacuated to dried lipid and is deposited on bottle wall.Stop the rotation after continuing suction more than 45min, from water-bath, mention round-bottomed flask, valve-off makes rotary evaporator and vacuum source isolate.Container is taken off from rotary evaporator.
3. vacuum drying: sealed by round-bottomed flask bottleneck preservative film, pricks hole some, puts into vacuum drying oven, closes blow vent, opens vacuum valve, vacuum pump, starts evacuation; After evacuation completes, close vacuum valve, vacuum pump.Vacuum drying starts, and sample is placed 30 ~ 40min.
4. aquation lipid: take out flask after removing vacuum from exsiccator, add 5ml (NH
4)
2sO
4solution aquation dry mixed lipid membrane.Ultrasonic aquation in 60 DEG C of water-baths, rotation round-bottomed flask peels off completely to the film on flask inwall and is mixed in (NH
4)
2sO
4in solution.And then the single dispersing body that ultrasonic 30min becomes clarification to mixture in water-bath or have an opalescence homodisperse colloid solution.
5. filter: use squeezer, the filter membrane choosing 200nm filters, and each pipette samples filters 11 times.
6. dialyse, hatch: dialyse 10 hours, within each hour, change once peripheral NaCl solution.Get the liposome after 3ml dialysis after end, add 225 μ l Doxorubicin solution, at 50 DEG C, hatch 30min.
Before the liposome making step of not bag medicine, 3 steps are identical, the 4th step 1.25mM tris-HCl 5.6ml aquation.
Embodiment 2
Lipopeptid structure is [CH
3-(CH
2)
4-VAQLEVK-VAQLESK-VSKLESK-VSSLESK] the preparation of lipopeptid-liposome
the preparation of solution
1. the preparation of phospholipid solution: use analytical balance to weigh 11.52mg DPPC, 12.4mg DSPC and 1.09mgDPPE, be added in 5ml volumetric flask together, dissolves standardize solution with the mixed solvent that Lv Fang ︰ methanol is 4 ︰ 1.
2. the preparation of cholesterol solution: use analytical balance to take 2.5mg cholesterol, the mixed solvent being 4 ︰ 1 with Lv Fang ︰ methanol dissolves and is settled in 5ml volumetric flask.
3. the preparation of lipopeptide solution: use analytical balance to take 0.5mg leucine zipper type lipopeptid CH
3-(CH
2)
4-VAQLEVK-VAQLESK-VSKLESK-VSSLESK is that dissolution with solvents is settled in 5ml volumetric flask with methanol.
4. the preparation of rhodamine liquor: get 20mg rhodamine in 10ml volumetric flask, with the Tris-HCl buffer solution standardize solution prepared, obtains 2mg/ml rhodamine liquor.Process for preparation notes lucifuge, prevents rhodamine liquor fluorescent quenching.
5. ammonium sulfate, NaCl solution, the preparation of dextran solution is the same.
the preparation of liposome
This experiment adopts thin film aquation legal system for liposome.
1. the preparation of mixed solution: get phospholipid solution 1.5ml, cholesterol solution 1.82ml, lipopeptide solution all adds, then adds 8ml chloroform, operates, obtain mixed solution with embodiment 1.
2. revolve steaming: immersed by flask in 45 DEG C of waters bath with thermostatic control, rotary speed is set to 106r/min, evacuation is until dried lipid is deposited on bottle wall.Stop the rotation after continuing suction more than 45min, from water-bath, mention round-bottomed flask, valve-off makes rotary evaporator and vacuum source isolate.Container is taken off from rotary evaporator.Vacuum drying 30min.
3. aquation lipid: add 5ml (NH in round-bottomed flask
4)
2sO
4solution, aquation mixing lipid membrane.Ultrasonic aquation in 55 DEG C of water-baths, rotation round-bottomed flask peels off completely to the film on flask inwall and is mixed in (NH
4)
2sO
4in.Then the single dispersing body that ultrasonic 30min becomes clarification to mixture in water-bath or have an opalescence homodisperse colloid solution.
4. filter: use squeezer, the filter membrane choosing 200nm filters, and each pipette samples filters 11 times.
5. dialyse, hatch: dialyse 16 hours, within each hour, change once peripheral NaCl solution.Get the liposome after 2ml dialysis after end, add 150 μ l rhodamine liquor, at 50 DEG C, hatch 60min.
6. cross pillar: the liposome of parcel rhodamine is crossed glucosan pillar, and the rhodamine do not wrapped up is absorbed by glucosan, and the rhodamine being wrapped in liposome interior is not then adsorbed.
Embodiment 3
Lipopeptid structure is [CH
3-(CH
2)
4-VAQLEVK-VAQLESK-VSKLESK-VSSLESK] the preparation of lipopeptid-liposome
the preparation of solution
1. the preparation of phospholipid solution: use analytical balance to weigh 11.52mg DPPC, 12.4mg DSPC and 1.09mg DPPE, be added in 5ml volumetric flask together, dissolves standardize solution with the mixed solvent that Lv Fang ︰ methanol is 4 ︰ 1.
2. the preparation of cholesterol solution: use analytical balance to take 2.5mg cholesterol, the mixed solvent being 4 ︰ 1 with Lv Fang ︰ methanol dissolves and is settled in 5ml volumetric flask.
3. the preparation of lipopeptide solution: use analytical balance to take 0.5mg leucine zipper type lipopeptid CH
3-(CH
2)
4-VAQLEVK-VAQLESK-VSKLESK-VSSLESK is that dissolution with solvents is settled in 5ml volumetric flask with methanol.
4. the preparation of rhodamine liquor: get 20mg rhodamine in 10ml volumetric flask, with the Tris-HCl buffer solution standardize solution prepared, obtains 2mg/ml rhodamine liquor.Process for preparation notes lucifuge, prevents rhodamine liquor fluorescent quenching.
the preparation of liposome
This experiment adopts thin film aquation legal system for liposome.
1. the preparation of mixed solution: get phospholipid solution 1.5ml, cholesterol solution 1.82ml, lipopeptide solution all adds, then adds 8ml chloroform, operates, obtain mixed solution with embodiment 1.
2. revolve steaming: immersed by flask in 45 DEG C of waters bath with thermostatic control, rotary speed is set to 106r/min, evacuation is until dried lipid is deposited on bottle wall.Stop the rotation after continuing suction more than 45min, from water-bath, mention round-bottomed flask, valve-off makes rotary evaporator and vacuum source isolate.Container is taken off from rotary evaporator.Vacuum drying 30min.
3. aquation lipid: add 5.6ml rhodamine liquor in round-bottomed flask, aquation mixing lipid membrane.Ultrasonic aquation in 50 DEG C of water-baths, rotation round-bottomed flask peels off completely to the film on flask inwall and is mixed in rhodamine liquor.
4. filter: use squeezer, the filter membrane choosing 200nm filters, and each pipette samples filters 11 times.
5. cross pillar: the liposome of parcel rhodamine is crossed glucosan pillar, and the rhodamine do not wrapped up is absorbed by glucosan, and the rhodamine being wrapped in liposome interior is not then adsorbed.
Embodiment 4
Lipopeptid structure is [CH
3-(CH
2)
5-VSSLESK-VSSLESK-VSSLESK] the preparation of lipopeptid-liposome
1. the preparation of phospholipid solution: use analytical balance to weigh 5.79mg DPPC, 6.34mg DSPC and 0.55mg DPPE, being added to together in the volumetric flask of 5ml, is the mixed proportion dissolution with solvents standardize solution of 4 ︰ 1 with Lv Fang ︰ methanol.
2. the preparation of cholesterol solution: use analytical balance to take the cholesterol of 2.5mg, be settled in 10ml volumetric flask with the mixed proportion dissolution with solvents that Lv Fang ︰ methanol is 4 ︰ 1.
3. the preparation of lipopeptide solution: use analytical balance to take 0.5mg leucine zipper type lipopeptid CH
3-(CH
2)
5-VSSLESK-VSSLESK-VSSLESK is that dissolution with solvents is settled in 5ml volumetric flask with methanol.
The preparation of 4.tris-HCL buffer solution is the same.
5. the preparation of uranine yellow solution: get 20mg uranine yellow and be dissolved in 10ml volumetric flask, with the tris buffer solution standardize solution prepared, obtains 2mg/ml uranine yellow solution.Process for preparation notes lucifuge, prevents uranine yellow from seeing photolysis.
the preparation of liposome
1. the preparation of mixed solution: get phospholipid solution 1.5ml, cholesterol solution 1.82ml, lipopeptide solution 0.236ml, then add 8ml chloroform, operate with embodiment 1, obtain mixed solution.
2. revolve steaming: immersed by flask in 45 DEG C of waters bath with thermostatic control, rotary speed is set to 106r/min evacuation until dried lipid is deposited on bottle wall.Stop the rotation after continuing suction more than 45min, from water-bath, mention round-bottomed flask, valve-off makes rotary evaporator and vacuum source isolate.Container is taken off from rotary evaporator.
3. aquation lipid: add 5.6ml uranine yellow solution in round-bottomed flask, aquation mixing lipid membrane.Ultrasonic aquation 30min in 55 DEG C of water-baths, rotation round-bottomed flask peels off completely to the film on flask inwall and is mixed in uranine yellow.
4. filter: use squeezer, the filter membrane choosing 200nm filters, and each pipette samples filters 11 times.
5. cross pillar: the liposome of parcel uranine yellow is crossed glucosan pillar, and the uranine yellow do not wrapped up is absorbed by glucosan, and the uranine yellow being wrapped in liposome interior is not then adsorbed.
Embodiment 5
Lipopeptid-liposome medicine carrying release
Lipopeptid structure is [CH
3-(CH
2)
5-VSSLESK-VSSLESK-VSSLESK] the preparation of lipopeptid-liposome
1. the preparation of phospholipid solution: use analytical balance to weigh 5.79mg DPPC, 6.34mg DSPC and 0.55mgDPPE, being added in the volumetric flask of 5ml together, is the mixed proportion dissolution with solvents standardize solution of 4 ︰ 1 with Lv Fang ︰ methanol.
2. the preparation of cholesterol solution: use analytical balance to take the cholesterol of 2.5mg, be settled in 10ml volumetric flask with the mixed proportion dissolution with solvents that Lv Fang ︰ methanol is 4 ︰ 1.
3. the preparation of lipopeptide solution: use analytical balance to take 0.5mg leucine zipper type lipopeptid CH
3-(CH
2)
5-VSSLESK-VSSLESK-VSSLESK is that dissolution with solvents is settled in 5ml volumetric flask with methanol.
4. the preparation of ammonium sulfate, NaCl solution, dextran solution is the same.
5. the preparation of uranine yellow solution: get 20mg uranine yellow and be dissolved in 10ml volumetric flask, with the tris buffer solution standardize solution prepared, obtains 2mg/ml uranine yellow solution.Process for preparation notes lucifuge, prevents uranine yellow from seeing photolysis.
the preparation of liposome
1. the preparation of mixed solution: get phospholipid solution 1.5ml, cholesterol solution 1.82ml, lipopeptide solution 0.236ml, then add 8ml chloroform, operate with embodiment 1, obtain mixed solution.
2. revolve steaming: immersed by flask in 45 DEG C of waters bath with thermostatic control, rotary speed is set to 106r/min evacuation until dried lipid is deposited on bottle wall.Stop the rotation after continuing suction more than 45min, from water-bath, mention round-bottomed flask, valve-off makes rotary evaporator and vacuum source isolate.Container is taken off from rotary evaporator.
3. aquation lipid: add 5ml (NH in round-bottomed flask
4)
2sO
4solution, aquation mixing lipid membrane.Ultrasonic aquation 30min in 55 DEG C of water-baths, rotation round-bottomed flask peels off completely to the film on flask inwall and is mixed in (NH
4)
2sO
4in.
4. filter: use squeezer, the filter membrane choosing 200nm filters, and each pipette samples filters 11 times.
5. dialyse, hatch: dialyse 12 hours, within each hour, change once peripheral NaCl solution.Get the liposome after 3ml dialysis after end, add 225 μ l uranine yellow solution, at 50 DEG C, hatch 30min.
6. cross pillar: the liposome of parcel uranine yellow is crossed glucosan pillar, and the uranine yellow do not wrapped up is absorbed by glucosan, and the uranine yellow being wrapped in liposome interior is not then adsorbed.
List of references:
[1] Koning, G.A.; Eggermont, A.M.M.; Lindner, L.H.; Ten Hagen, T.L.M. are used for the responsive to temperature of chemotherapy of tumors, high thermotherapy type liposome .Pharm.Res.2010,27,1750 – 1754.
[2] Needham, D.; Anyarambhatla, G.; Kong, G.; Dewhirst, M.W. are used for the Novel temperature-sensitive liposome of hyperthermia treatment: characteristic and the test .Cancer Res.2000 in transplanted tumor, 60,1197 – 1201.
[3] Kono, K.; Murakami, T.; Yoshida, T.; Haba, Y.; Kanaoka, S.; Takagishi, T.; Aoshima, S. are containing the liposome of temperature sensitive type block polymer: copolymer chain length affect .Bioconjugate Chem.2005,16,1367 – 1374.
Claims (10)
1. have a lipopeptid for temperature sensitivity, its molecular structure is CH
3-(CH
2) n-(VXXLXXX) m, wherein n:4 ~ 10, m:3 ~ 5, V: valine; L: leucine, X: except the hydrophilic amino acid except valine and leucine.
2. there is lipopeptid-liposome of temperature sensitivity, be made up of with the mol ratio self assembly of 1 ~ 50:200 lipopeptid described in claim 1 and phospholipid.
3. lipopeptid-liposome as claimed in claim 2, wherein said phospholipid is selected from HSPC, DOPC, DPPE, DPPC and DSPG.
4. lipopeptid-liposome as claimed in claim 2, described lipopeptid-liposome is the liposome of not bag medicine, and it disperses aquation legal system to obtain by thin film, comprising:
1) lipopeptid and phospholipid are dissolved in the organic solvent being selected from chloroform, absolute methanol and ethanol respectively, in 30-60 DEG C of outstanding steaming 30-60min after gained solution fully mixes, obtain lipopeptid-liposome membrane;
2) solvent evaporate off in vacuo, continue to drain after lipopeptid-liposome deposition bottle wall, with tris-HCl solubilize lipopeptid-liposome membrane, 30-60 DEG C of ultrasonic disperse 10-30min, obtains lipopeptid-liposome solutions;
3) with filter by step 2) lipopeptid-liposome solutions of gained filters by the extruder of filter membrane diameter 200nm.
5. lipopeptid-liposome as claimed in claim 2, described lipopeptid-liposome is the liposome of bag medicine, and it disperses aquation legal system to obtain by thin film, comprising:
1) lipopeptid and phospholipid are dissolved in the organic solvent being selected from chloroform, absolute methanol and ethanol respectively, in 30-60 DEG C of outstanding steaming 30-60min after gained solution fully mixes, obtain lipopeptid-liposome membrane;
2) solvent evaporate off in vacuo, continues to drain after lipopeptid-liposome deposition bottle wall, adds 5ml (NH
4)
2sO
4solution aquation lipopeptid-liposome membrane, 30-60 DEG C of ultrasonic disperse 10-30min, obtains lipopeptid-liposome solutions;
3) with filter by step 2) lipopeptid-liposome solutions of gained filters by the extruder of filter membrane diameter 200nm.
6. lipopeptid-the liposome as described in claim 4 or 5, also adds the cholesterol being dissolved in organic solvent when wherein said lipopeptid and phospholipid mixing and is mixed together.
7. lipopeptid-liposome as claimed in claim 6, wherein said cholesterol is catatonic cholesterol.
8. lipopeptid-liposome as claimed in claim 5, described liposome is the drug-loaded liposome having wrapped up medicine, and it obtains by the following method:
1) by liposome described in claim 5 after dialysis, be placed in 40 DEG C ~ 60 DEG C water-baths, with medicine: the ratio of liposome=1:20 drips drug solution, hatches 30min ~ 40min in a water bath after being added dropwise to complete, every 10min concussion mixing once;
2) non-encapsulated medicine is separated by dextran microgel column.
9. lipopeptid-liposome as claimed in claim 8, solvent evaporate off in vacuo described in its preparation method is rotary evaporation in 45 DEG C of waters bath with thermostatic control, described in add (NH
4)
2sO
4first ultrasonic preheating in 60 DEG C of water-baths before solution aquation, described ultrasonic disperse carries out 30min.
10. lipopeptid-liposome described in claim 2 is preparing the purposes on targeted drug.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105664171A (en) * | 2016-01-05 | 2016-06-15 | 清华大学 | Immunoregulator, and preparation method and application thereof |
| CN110693836A (en) * | 2019-10-21 | 2020-01-17 | 中国石油大学(华东) | Lipopeptide self-assembly ultra-small nano vesicle and preparation method thereof |
| CN111000802A (en) * | 2019-12-27 | 2020-04-14 | 中山大学 | Antibacterial peptide liposome preparation and preparation method thereof |
| CN112062816A (en) * | 2020-09-21 | 2020-12-11 | 河南中医药大学 | Polypeptide with pH sensitivity, polypeptide-polymer conjugate, preparation method and application |
| CN113117094A (en) * | 2021-04-06 | 2021-07-16 | 华东理工大学 | Temperature-sensitive polypeptide-encapsulated mesoporous silica intelligent drug carrier and preparation thereof |
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| WO1999065466A1 (en) * | 1998-06-18 | 1999-12-23 | Duke University | Temperature-sensitive liposomal formulation |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105664171A (en) * | 2016-01-05 | 2016-06-15 | 清华大学 | Immunoregulator, and preparation method and application thereof |
| CN105664171B (en) * | 2016-01-05 | 2019-02-05 | 清华大学 | Immunomodulator, preparation method and application |
| CN110693836A (en) * | 2019-10-21 | 2020-01-17 | 中国石油大学(华东) | Lipopeptide self-assembly ultra-small nano vesicle and preparation method thereof |
| CN111000802A (en) * | 2019-12-27 | 2020-04-14 | 中山大学 | Antibacterial peptide liposome preparation and preparation method thereof |
| CN112062816A (en) * | 2020-09-21 | 2020-12-11 | 河南中医药大学 | Polypeptide with pH sensitivity, polypeptide-polymer conjugate, preparation method and application |
| CN113117094A (en) * | 2021-04-06 | 2021-07-16 | 华东理工大学 | Temperature-sensitive polypeptide-encapsulated mesoporous silica intelligent drug carrier and preparation thereof |
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