CN103073646A - Polypeptide carrying melittin, nano-particles carrying melittin, and applications thereof - Google Patents
Polypeptide carrying melittin, nano-particles carrying melittin, and applications thereof Download PDFInfo
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Abstract
The invention discloses a polypeptide carrying melittin, nano-particles carrying melittin, and applications thereof. The invention belongs to the field of biological science and drug carriers. The peptide is composed of alpha helical polypeptide, connection sequence, and melittin which are connected in series with a covalent bond form. The nano-particles are formed through three effective bonding manners of polypeptides, phospholipids, and cholesterol esters carrying melittin. With the nano-particles prepared from the polypeptide carrying melittin, toxic and side effects of melittin against bodies can be effectively reduced, and effects can be performed specifically at diseased areas such as tumors. The nano-particles can be used in clinical treatments.
Description
Technical field
The invention belongs to bio-science and pharmaceutical carrier field, particularly nano particle and the application thereof of a kind of polypeptide that delivers mellitin, delivery mellitin.
Background technology
Mellitin (melittin, GIGAVLKVLTTGLPALISWIKRKRQQ) is a kind of polypeptide of amphipathic αhelix, is the main active ingredient in the bee venom, accounts for the 40%-60% of its dry weight.The activity of mellitin is very high, has the antibacterial of wide spectrum, can bring into play its effect within the extremely short time, and its efficient is common antibiotic hundreds of times.Mellitin has destructive very efficiently for membrane structure, comprise the membrane structure of some organoids in plasma membrane and the born of the same parents.Its mechanism of action mainly contains by its amphipathic structure and is embedded in the membrane structure, then punches at film, causes membrane structure to be broken, and the variation of film internal and external environment finally causes the death of cell; Also can pass through to affect the key protein in the many signal paths that mediate tumor proliferation in the cell, and then cause apoptosis.Therefore, mellitin can brought into play very large effect aspect the treatment tumour.In addition, correlative study shows that the activity of expression that mellitin can also be by suppressing the HIV-1 gene and LTR suppresses the propagation of hiv virus; The effect that also has relieving inflammation and relaxing pain, analgesia intensity is 40% of morphine, and analgesia time is lasting, and anti-inflammatory activity is more than 100 times of nearly hydrocortisone, can not cause immunosuppressant effect; Mellitin also can be regulated the release of adrenocortical hormone, reaches the effect of rheumatism, rheumatoid arthritis; In addition, mellitin can also cause neuroendocrine response, enhancing body radiation resistance and prevent platelet aggregation, antithrombotic effect.In a word, mellitin all is with a wide range of applications on the treatment various diseases.
Yet, because the characteristic of mellitin self is difficult to it is directly applied in the disease treatment of live body.Because mellitin meeting after injecting blood vessel is rapid and erythrocyte binding, destroys cytolemma, so that produce strong hemolytic reaction.And directly mellitin is applied to the live body treatment, and may damage organism normal cell, bring great toxic side effect.In addition, the transformation period of mellitin in body is very short, and accretion rate is very fast, and is unfavorable for clinical disease treatment.
The nanometer vehicle is paid close attention to by people just gradually as a kind of novel administering mode.Utilizing nano particle delivery mellitin is a kind of method of effectively avoiding its toxic side effect.Yet, use at present the report of nano particle delivery mellitin actually rare.Some researcher attempts using liposome (liposome) that mellitin is delivered, and the Electronic Speculum result shows and loads that great change can occur the structure of liposome behind the mellitin, even some can be degraded.Although the dish type particle that the PEG that has on this basis the scholar to propose more stable use liposomal delivery mellitin modifies really is not applied to the live body treatment.Also having the scholar to use PLGA[poly (D, L-lactide-co-glycolide acid)] particle has been realized the delivery to the high encapsulation rate of mellitin, but they have equally just carried out external contrast experiment, do not continue deeply.Some other nano particle of delivery mellitin is same to be limited greatly because its haemolysis character can not get improving.At present, there be a kind of can in living animal, stablizing based on perfluoro-carbon (perfluocarbon, PFC) nano particle to transport mellitin, when having reduced its toxic side effect, can effectively bring into play antitumor action.In addition, this mellitin nano particle can also be realized the specific enrichment of tumour after having modified the target group.But this class PFC grain diameter is larger, between 200-300nm, can't pass through the slit (20-40nm) of netted collegen filament fine and close in the solid tumor, be difficult to effectively be diffused into the inside of tumour, especially to the not abundant solid tumor of blood vessel, be difficult to reach the result for the treatment of of expection.And metabolism, drainage and the bio-toxicity of PFC particle in human body also needs the further experiment checking; As a kind of greenhouse gases material, the extensive application of perfluoro-carbon also can bring the destruction of environment in addition.
Therefore, develop a kind of ultra-small diameter grain (nano particle medicine-carrying method of＜40nm) stable delivery mellitin, effectively avoid the hemolytic of mellitin and to Normocellular toxic side effect, have simultaneously specific enrichment in lesion region and effective ability that discharges mellitin, will greatly promote the application of mellitin in clinical disease treatment.
Summary of the invention
The objective of the invention is provides a kind of polypeptide and nano particle, preparation method and application thereof that delivers mellitin for addressing the above problem.The nano particle that the polypeptide preparation of utilization delivery mellitin obtains can effectively reduce mellitin for the toxic side effect of body, becomes the zone in diseases such as tumours specifically and plays a role, and can be applied to clinical treatment.
The technical solution adopted in the present invention is:
A kind of polypeptide that delivers mellitin, described polypeptide are to be connected in series by α spiral polypeptide, catenation sequence and the mellitin form with covalent linkage.
Preferably, the aminoacid sequence of described α spiral polypeptide is DWFKAFYDKVAEKFKEAF.
Preferably, the aminoacid sequence of described catenation sequence is GSG.
A kind of nano particle that delivers mellitin, described nano particle requires polypeptide, the phosphatide of 1 described delivery mellitin, the mode of three kinds of combinations of cholesterol ester to form by claim.
Preferably, described phosphatide is DMPC(1,2-dimyristoyl-sn-glycero-3-phosphocholine).
A kind of application that delivers the nano particle of mellitin, described nano particle can effectively reduce mellitin for the toxic side effect of body, become the zone in the disease that comprises tumour specifically and play a role, and can be applied to clinical treatment.
The present invention has the following advantages:
1) physicochemical property is good: about utilizing median size that the dynamic laser light scattering method records nano particle for 12nm, Electronic Speculum result shows, the uniform particle diameter of nano particle, good dispersity are without clustering phenomena.
2) good biocompatibility: prepare the polypeptide that the employed raw material of this nano particle is phosphatide, cholesterol ester and delivery mellitin, these starting material all are used for clinical or clinical trial separately, have good biocompatibility.
3) preparation technology is simple, is convenient to large-scale production.
4) entrapment efficiency meets the Pharmacopoeia of the People's Republic of China for the requirement of microcapsule formulation greater than 80%; And than free mellitin, adopt the nano particle entrapment efficiency of the delivery mellitin of the polypeptide preparation that delivers mellitin to improve 18 times.
5) result for the treatment of is good: take solid tumor as example, the nano particle of delivery mellitin can be by EPR effect (enhanced permeability and retention effect) section's enrichment within it of solid tumor, the local drug concentration of Effective Raise tumor region, the netted collagen slit that its extra small nano-scale can freely pass the solid tumor densification arrives tumour core position, thereby killing tumor cell efficiently, but also can effectively avoid the resistance of tumour cell.
6) toxic side effect is low: the polypeptide design of delivery mellitin can increase the interaction between itself and the phosphatide, it is imbedded in the middle of the individual layer phospholipid layer, thereby has avoided directly contacting with Normocellular with hemocyte.Results of Animal shows, the nano particle treatment group of delivery mellitin can suppress the growth of tumour significantly, and between the blood parameters of experiment mice and the control group without significant difference and all in range of normal value.
7) multifunction: the nano particle of delivery mellitin is except directly applying to the disease treatments such as tumour, rheumatism, thrombus, can also load dye molecule and the drug molecule that is used for disease treatment in its core, also can load simultaneously targeted polypeptide or the therapeutical peptide of other type, realize the collaborative target of disease or the effect of Synergistic treatment.
Description of drawings
The present invention is further detailed explanation below in conjunction with drawings and embodiments.
Fig. 1 is FPLC(Fast protein liquid chromatography) two waveband absorption-time plot during the nano particle of the delivery mellitin of the low dose of preparation of system purifying;
Two waveband absorption-time plot when Fig. 2 is the nano particle of FPLC system purifying delivery mellitin;
Two waveband absorption-time plot when Fig. 3 is the nano particle of delivery mellitin of the heavy dose of preparation of FPLC system purifying;
Triband absorption-time plot when Fig. 4 is the nano particle of the FPLC system purifying core delivery mellitin that loads fluorescence dye DiR-BOA;
Triband absorption-time plot when Fig. 5 is the nano particle of the FPLC system purifying core delivery mellitin that loads Fluo-BOA;
Fig. 6 is for using dynamic laser scattering of light (DLS) system testing to deliver the nanometer particle size result of the nano particle of mellitin;
Fig. 7 is the transmission electron microscope image of the nano particle of delivery mellitin;
Fig. 8 is that the nano particle of delivery mellitin and the erythrocyte hemolysis test of free mellitin are compared;
Fig. 9 is the propagation inhibition ability comparison for various tumour cells of nano particle and the free mellitin of delivery mellitin;
Figure 10 suppresses the B16 tumour at the time meta-gross tumor volume graphic representation of C57BL/6 mouse tumor growth for the nano particle of delivery mellitin;
Figure 11 suppresses the tumor mass pictorial diagram of B 16 tumours after C57BL/6 mouse interior therapeutic was peeled off in treatment group and the control group mice body in the time of the 13rd day for the nano particle of delivery mellitin;
Figure 12 is blood biochemical and the hemocyte index of mouse model in the time of the 13rd day of the nano particle treatment lotus B16 tumour of delivery mellitin.
Embodiment
The polypeptide of delivery mellitin, this peptide is to be connected in series by α spiral polypeptide, catenation sequence and the mellitin form with covalent linkage.The aminoacid sequence of α spiral polypeptide is in the present embodiment: DWFKAFYDKVAEKFKEAF, the aminoacid sequence of catenation sequence are GSG, and the aminoacid sequence of mellitin is: GIGAVLKVLTTGLPALISWIKRKRQQ.The aminoacid sequence of the polypeptide of this delivery mellitin is that SEQ ID NO.1 is described in the sequence table.
The nano particle of preparation delivery mellitin the steps include:
1) with 3 μ mol DMPC
(1,2-dimyristoyl-sn-glycero-3-phosphocholine), the chloroformic solution of 0.2 μ mol cholesterol ester (Cholesteryl oleate, be called for short C.O) fully mixes in glass test tube, and with sealed membrane the test tube mouth is shut;
2) in stable nitrogen gas stream, invisible spectro chloroform is dried up, make the mixture in the step 1) form thin film in the test tube bottom;
3) test tube is put into vacuum drier vacuum-drying 1h;
4) add the phosphoric acid buffer of 1ml in the test tube, be filled with nitrogen-sealed after, utilize vortex concussion instrument with the uniform milky suspension liquid of the abundant resuspended formation of medicine film of test tube bottom;
5) with 48 ℃ of ultrasonic 1h of test tube;
6) use syringe to add the PBS solution of the polypeptide that contains 0.19 μ mol delivery mellitin in the test tube of sealing, seal behind the mixing, 4 ℃ of placements are spent the night.
7) next day, use FPLC system purifying, collect effective solution concentrated for subsequent use.
The nano particle of delivery mellitin utilizes above-mentioned steps to synthesize, and uses the FPLC system that the nano particle of the delivery mellitin for preparing is carried out purifying, and its result is with reference to such as Fig. 1 to Fig. 3.Two waveband absorption-time plot when Fig. 1 is the nano particle of delivery mellitin of the low dose of preparation of FPLC system purifying.Use the polypeptide of 0.19 μ mol delivery mellitin to prepare in a small amount the nano particle that delivers mellitin.The 215nm absorption curve value that shows among Fig. 1 is 1/5 of actual value.Occur altogether 3 peaks among Fig. 1, be defined as respectively Peak 1,2,3 according to time sequence; Wherein Peak 2 is the nano particle of delivery mellitin, and Peak 3 is free polypeptide.The integral area of Peak 2 accounts for 80.3% of the total area, and free content of peptides (Peak 3 areas) is 8.74%.Two waveband absorption-time plot when Fig. 2 is FPLC system purifying mellitin nano particle.Use simultaneously 0.19 μ mol alpha-helix polypeptide and 0.19 μ mol mellitin to prepare in a small amount the mellitin nano particle.The 215nm absorption curve value that shows among the figure is 1/5 of actual value.The area of Peak 2 among Fig. 2 is 4.22%, and free content of peptides is 88.28%.Experimental result shows, uses the method for tandem polypeptide can make the delivery rate of mellitin improve 18 times, and the utilization ratio of Effective Raise polypeptide.Two waveband absorption-time plot when Fig. 3 is the nano particle of delivery mellitin of the heavy dose of preparation of FPLC system purifying.Use the polypeptide of 1.9 μ mol delivery mellitin to prepare in a large number the nano particle that delivers mellitin.Amplify on year-on-year basis 10 times with the preparation dosage shown in Fig. 1, the 215nm absorption curve value that shows among the figure is 1/5 of actual value.）。Peak 2 areas among Fig. 3 are 79.5%, and are similar with the productive rate of in a small amount preparation, show that the method productive rate in amplification process does not obviously reduce, and can carry out large-scale preparation and purification.
As the expansion of the nano particle that delivers mellitin, also can in its core, load fluorescence dye in the preparation.Proportioning raw materials is: 3 μ mol DMPC, 0.1 μ mol C.O, 0.3 μ mol DiR-BOA(near infrared fluorescent dye through modifying, excite with emission wavelength and be respectively 748nm and 780nm), the polypeptide of 0.57 μ mol delivery mellitin, its FPLC purification result is referring to Fig. 4, and the 215nm absorption curve value that shows among Fig. 4 is 1/10 of initial value.3 μ mol DMPC, 0.1 μ mol C.O, 0.3 μ mol Fluo-BOA(green fluorescence dyestuff through modifying, excite with emission wavelength and be respectively 495nm and 525nm), 0.38 μ mol α-melittin, its FPLC purification result is participated in Fig. 5, and the 215nm absorption curve value that shows among Fig. 5 is 1/10 of initial value
The mensuration of the nano particle basic nature matter of delivery mellitin: the measurement of nanometer particle size shows d=11.76 ± 2.40nm, and its result is referring to Fig. 6.The transmission electron microscope photo is referring to Fig. 7, and the nano particle that demonstrates the delivery mellitin among Fig. 7 is the nano particle of a kind of monodispersity, uniform particle diameter, does not have obvious clustering phenomena.
The nano particle of the delivery mellitin for preparing among the embodiment 1 and free mellitin for the comparison of hemolysis referring to Fig. 8.The nano particle that shows the delivery mellitin among Fig. 8 reaches in the concentration of mellitin that hemolysis rate does not still reach 10% in the situation of 60 μ M, free mellitin then at 1 μ M near 100%.The nano particle of proof delivery mellitin can effectively reduce the hemolysis of mellitin, can not destroy red corpuscle after the intravenous injection, provides the foundation for it is applied to the live body treatment.
The nano particle of delivery mellitin and the comparison of free mellitin aspect the cell killing ability are with reference to Fig. 9.The nano particle that relatively can find out the delivery mellitin by Fig. 9 and cell proliferation experiment can reduce mellitin for the destruction of cell; and then can in the vivo applications process, protect organism normal cell to avoid infringement, effectively reduce the toxic side effect that mellitin brings.
The nano particle of the delivery mellitin for preparing among the embodiment 1 is treated tumor model at body:
Make up B16 Subcutaneous tumor model.Digestion B16 cell carries out cell counting after using twice of the PBS rinsing of sterilization, at last cell concn is decided to be 8 * 10
6Individual/L.Anesthesia C57BL/6 mouse, the tumour cell injection of solution is subcutaneous to the left leg root of mouse, and volume injected is 100 μ l.The date is fixed for the 0th day in inoculation, and the date is thereafter remembered respectively and does the 1st, 2,3 ... my god.According to demand mouse was carried out the stratified random grouping at the 4th day, every establishment view quantity is not less than 5.
In the time of the 5th day, begin tail vein injection.First each mouse is carried out mark before the injection, take by weighing the quality of each mouse, record in the form that pre-establishes.
Only need the PBS of tail vein injection sterilization to get final product during the injection of Control group, injection volume is 0.2ml/10g.
The I.v.therapy group needs tail vein injection α-melittin-NP, and injected dose is 20mg/kg (content of peptides), and volume injected is 0.2ml/10g.
The I.p.therapy group needs abdominal injection α-melittin-NP, and injected dose is 20mg/kg (content of peptides), and volume injected is 0.3ml/10g.
NP Control group is as the empty carrier contrast, and the concentration of injection determines according to phospholipid concentration, and phospholipid concentration need to be consistent with the phospholipid concentration of Therapy group.
Injection cycle is for every other day injecting once totally 4 times.Blood extracting assay and put to death immediately mouse and carry out subsequent experimental operation in the time of the 13rd day.Measure gross tumor volume with vernier callipers.Measured since the 5th day, every other day survey once, take off data need in time be recorded in the form.The calculation formula of volume is V=0.5 * L * W * H.
Through 4 inhibitory rate to 90% of taking turns the nano particle that shows the delivery mellitin after the treatment, compare with control group and to have significant result for the treatment of.This effect has also clearly been confirmed in tumor mass contrast after the dissection.Its result is referring to Figure 10 and Figure 11, and * represents P＜0.05 among Figure 10.
Detect Physiology and biochemistry level and the hemocyte index of the rear mouse of nano particle treatment of delivery mellitin, participate in Figure 12.Experimental result shows, the nano particle of delivery mellitin does not produce overt toxicity to the liver kidney of mouse, and blood cell parameter and contrast difference are little, confirm the biocompatibility that it is good.
Embodiment 4
The three class materials (phosphatide, cholesterol acid ester, polypeptide) of the nano particle of the preparation delivery mellitin for preparing among the embodiment 1 have all had application clinically.
Phospholipid substance has been applied to clinical treatment, and liposome is exactly a good example.Doxil is a kind of medicine that is used for treating Kaposis sarcoma with the liposome Zorubicin, and U.S. FDA has been ratified the treatment that Doxil is used for ovarian cancer and multiple myeloma.
Cholesterol is the material that itself exists in the human organism, has good biological safety.
Polypeptides matter also has many clinical treatments that are applied to, and gives an example: Lugua polypeptide is exactly a kind of medicine of good treatment fracture.Induce polypeptide class biotic factor can effectively promote to affect in the body bdgf synthetic of bone forming and absorption as the bone of this medicine main component.
The Lugua polypeptide injection liquid can obviously promote the formation of knitting and new bone, regulates bone metabolism, has the effects such as relieving inflammation and relaxing pain, and is to nonunion osteoporosis determined curative effect, without obvious adverse reaction, good to rheumatoid arthritis soft tissue injury curative effect.
Above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although with reference to preferred embodiment the present invention is had been described in detail, those of ordinary skill in the art is to be understood that, can make amendment or be equal to replacement technical scheme of the present invention, and not breaking away from the spirit and scope of technical solution of the present invention, it all should be encompassed in the middle of the claim scope of the present invention.
＜110〉Central China University of Science and Technology
＜120〉a kind of polypeptide and nano particle, preparation method and application thereof that delivers mellitin
<160> 1
<210> 1
<211> 47
<212> PRT
＜213〉artificial sequence
<400> 1
DWFKAFYDKVAEKFKEAFGSGGIGAVLKVLTTGLPALISWIKRKRQQ 47
Claims (6)
1. a polypeptide that delivers mellitin is characterized in that, described polypeptide is to be connected in series by α spiral polypeptide, catenation sequence and the mellitin form with covalent linkage.
2. the polypeptide of delivery mellitin according to claim 1 is characterized in that, the aminoacid sequence of described α spiral polypeptide is DWFKAFYDKVAEKFKEAF.
3. the polypeptide of delivery mellitin according to claim 1 is characterized in that, the aminoacid sequence of described catenation sequence is GSG.
4. a nano particle that delivers mellitin is characterized in that, described nano particle requires polypeptide, the phosphatide of 1 described delivery mellitin, the mode of three kinds of combinations of cholesterol ester to form by claim.
5. the nano particle of delivery mellitin according to claim 4 is characterized in that, described phosphatide is DMPC(1,2-dimyristoyl-sn-glycero-3-phosphocholine).
6. an application that delivers the nano particle of mellitin is characterized in that, described nano particle can effectively reduce mellitin for the toxic side effect of body, becomes the zone in the disease that comprises tumour specifically and plays a role, and can be applied to clinical treatment.
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| CN2012100645642A CN103073646A (en) | 2012-03-13 | 2012-03-13 | Polypeptide carrying melittin, nano-particles carrying melittin, and applications thereof |
| PCT/CN2013/000279 WO2013135103A1 (en) | 2012-03-13 | 2013-03-13 | Polypeptide carrying melittin, nanoparticle carrying melittin and use thereof |
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Cited By (9)
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| CN104784677A (en) * | 2014-05-13 | 2015-07-22 | 东北师范大学 | Application of melittin in preparation of drug used for inhibiting invasion metastasis of breast cancer cells |
| CN106632688A (en) * | 2016-12-23 | 2017-05-10 | 华中科技大学同济医学院附属协和医院 | Polypeptide for modifying microbubbles and GBM-targeting (glioblastoma multiforme targeting) pharmaceutical preparation |
| CN106699896A (en) * | 2016-12-05 | 2017-05-24 | 华中科技大学同济医学院附属协和医院 | Tumor killing polypeptide capable of being self-assembled into hydrogel and application thereof |
| CN109513000A (en) * | 2019-01-14 | 2019-03-26 | 华中科技大学同济医学院附属协和医院 | It is a kind of deliver melittin photoactive nanoparticles support preparation method and application |
| CN109692327A (en) * | 2017-10-23 | 2019-04-30 | 华中科技大学 | A kind of application of the nano particle of the application and delivery melittin of melittin |
| CN109692326A (en) * | 2017-10-23 | 2019-04-30 | 华中科技大学 | A kind of application of bee venom lipidic nanoparticles |
| CN113499321A (en) * | 2021-06-10 | 2021-10-15 | 南方医科大学 | Micro motor carrier and preparation method and application thereof |
| CN113559241A (en) * | 2021-07-22 | 2021-10-29 | 上海市宝山区中西医结合医院(上海中医药大学附属曙光医院宝山分院) | Melittin lipid nanoparticle and preparation method and application thereof |
| CN114099692A (en) * | 2021-11-30 | 2022-03-01 | 西南大学 | Antibacterial peptide-cell membrane compound, preparation method and application |
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| CN106699896A (en) * | 2016-12-05 | 2017-05-24 | 华中科技大学同济医学院附属协和医院 | Tumor killing polypeptide capable of being self-assembled into hydrogel and application thereof |
| CN106699896B (en) * | 2016-12-05 | 2020-06-05 | 华中科技大学同济医学院附属协和医院 | Tumor killing polypeptide capable of self-assembling into hydrogel and application thereof |
| CN106632688A (en) * | 2016-12-23 | 2017-05-10 | 华中科技大学同济医学院附属协和医院 | Polypeptide for modifying microbubbles and GBM-targeting (glioblastoma multiforme targeting) pharmaceutical preparation |
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| CN109692327B (en) * | 2017-10-23 | 2022-04-08 | 华中科技大学 | Application of nano-particles for carrying melittin |
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| CN113499321A (en) * | 2021-06-10 | 2021-10-15 | 南方医科大学 | Micro motor carrier and preparation method and application thereof |
| CN113559241A (en) * | 2021-07-22 | 2021-10-29 | 上海市宝山区中西医结合医院(上海中医药大学附属曙光医院宝山分院) | Melittin lipid nanoparticle and preparation method and application thereof |
| CN114099692A (en) * | 2021-11-30 | 2022-03-01 | 西南大学 | Antibacterial peptide-cell membrane compound, preparation method and application |
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