CN101361712B - Blood vessel target liposome carrier mediated by fiber forming growth factor receptor and preparation method and use thereof - Google Patents
Blood vessel target liposome carrier mediated by fiber forming growth factor receptor and preparation method and use thereof Download PDFInfo
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- CN101361712B CN101361712B CN200810304731XA CN200810304731A CN101361712B CN 101361712 B CN101361712 B CN 101361712B CN 200810304731X A CN200810304731X A CN 200810304731XA CN 200810304731 A CN200810304731 A CN 200810304731A CN 101361712 B CN101361712 B CN 101361712B
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Abstract
The invention belongs to the biology medicine field, and relates to a fiber growth factor receptor (FGFR)-mediated vascular targeted liposome carrier, a preparation method thereof and an application thereof. The invention aims at solving the technical problem of proving a cationic liposome with high transfection efficiency and tumor vascular targeting. The targeted liposome is prepared by using the components with the following mixture ratios: the mixture ration of DOTAP, cholesterol and tb FGF polypeptide is: the mol ratio of the DOTAP to the cholesterol equals to 2:1-2, and the weight of the tb FGF polypeptide is 10-80 percent of the total weight of the DOTAP and the cholesterol. The liposome can be combined with the FGF receptor (FGFR) on the surface of tumor new-generated vascular cells particularly, and can realize the preparation method thereof and the application of tumor vascular targeting therapy by wrapping up various micromolecule anti-tumor drugs, therapy genes and adenoviral vectors carrying therapy genes.
Description
Technical field
The invention belongs to biomedicine field, be specifically related to target liposomes carrier and the method for preparing and the purposes of a kind of fibroblast growth factor receptor (FGFR) mediation.
Background technology
Nanotechnology is the new technique that is born phase late 1980s and emerges rapidly, with information science and life sciences and be called big pillar science of 21 centurys three.At present, world many countries is all listed research in nanotechnology in the state key development field.Nanometer biotechnology is one of research focus of nanotechnology; China has all started " nano biological and Medical Technology " great special project in " 15 " 863 Program and program for tackling key problems in science and technology and Eleventh Five-Year Plan 863,973 planning periods, aspect nano-drug preparation, has obtained a series of advanced persons' original achievement.
Nanotechnology is that the research size is in the physicochemical properties that material had of 0.1～100nm and the science of function, and through manipulating single atom directly, molecule is assembled and creating the research field of the material of specific function.And in pharmaceutics research, generally the particle size range with nanoparticle is decided to be 1～500nm.
Because the drug-carried nanometer volume is littler, therefore has the advantage that a lot of macroparticles do not possess.Nanoparticle gets in the body can reduce engulfing of human body reticuloendothelial system macrophage effectively; And can pass through intercellular substance; Can through the minimum blood capillary of human body and blood brain barrier and by cell tissue absorb drug-carried nanometer can control medicine at the target spot position sustained release; Reduce the drug dose medicament curative effect enhancement and reduce drug toxicity; Medicament carrier system can avoid the pharmaceutically active forfeiture to help the storage and the transportation of medicine simultaneously, and the gene nano-complex can improve gene transfection efficient, realize targeting in the body.Because the plurality of advantages of drug-carried nanometer makes it become a kind of up-and-coming novel pharmaceutical formulation.
Liposome as the important component part in the nano material has shown good prospect at drug delivery system; Because the biodegradability of preparation liposome material; Characteristics such as toxicity is little make it aspect drug conveying, have many superioritys: but slow releasing pharmaceutical, prolong drug action time; Through the ligand modified target organs that conducts drugs to of targeting such as antibody, folic acid; Can guarantee to reduce the dosage of administration under the pharmaceutically-active prerequisite, alleviate or avoid toxic and side effects; Improve stability of drug, be beneficial to storage; Improve the water solublity (like the liposome medicament of paclitaxel) of insoluble medicine.Liposome, cationic polymer micelle (PEI, PAGA, PEG-PLL, chitosan etc.) in gene therapy since have biological safety better, good stability, non-immunogenicity, quiding gene capacity carry out the transformation of targeting property greatly, easily, amplify advantages such as production and process stabilizing, production cost are low easily; More and more favored and paid attention to, particularly cationic-liposome (grain through≤100nm) be widely used in the transmission system of gene therapy.But common cationic-liposome greatest problem is that transfection efficiency is low, and targeting property is poor.Its main cause is that (1) gene import system efficient is lower, lacks targeting; (2) some gene therapy vectors insert at random and are incorporated on the host cell chromosome, have to cause the potential danger of inserting sudden change and malignant transformation of cells; (3) therapeutic gene that imports is lacked effectively regulation and control etc.The existence of these problems also is the main restricting factor of the effective and safe gene therapy scheme of development.Matter of utmost importance in the gene therapy key technology is therapeutic gene to be transported to specified target tissue (or target organ) by selectivity (or relative specificity), and makes it to get into target cell.
Based on above reason, a kind of novel target liposomes carrier of exploitation is badly in need of in this area, can be targeted to the purpose cell in order to mediation micromolecule antitumor drug, reduces its toxicity to normal structure; Simultaneously, also efficiently the mediated gene medicine improves transfection efficiency in the expression of target cell.
Summary of the invention
Technical problem to be solved by this invention provides a kind of novel tumor vascular targeted liposome vectors that has.
The technical scheme that solves technical problem of the present invention has provided a kind of target liposomes carrier.This target liposomes is prepared from the composition of following proportioning: the composition following proportioning is prepared from: DOTAP and cholesterol; Its proportioning is mol ratio DOTAP: cholesterol=2: 1～2; Also contain the tbFGF polypeptide, the weight content of tbFGF polypeptide is 10～80% of DOTAP and a cholesterol gross weight.
Preferably, above-mentioned target liposomes carrier is prepared from the composition of following proportioning: DOTAP: cholesterol=2: 1～2 (mol ratio), the weight content of tbFGF polypeptide are 30～70% of DOTAP and cholesterol gross weights.
Wherein, the above-mentioned structure transformation to fibroblast growth factor (bFGF) that is meant obtains the bFGF peptide section of truncate, keeps the fragment of its bFGF and its receptors bind, and has lacked the bFGF polypeptide of the active peptide section that stimulates further signal conduction.Mainly be disappearance part heparin binding site, make it lack the activity that stimulates further signal conduction, the propagation that can not stimulate target cell.
Preferably, above-mentioned tbFGF comprises the 30-115 aminoacid of bFGF, and it has the aminoacid sequence of (Seq ID No.1) as follows:
krlycknggfflrihpdgrvdgvreksdphiklqlqaeergvvsikgvcanrylamkedgrllaskcvtdecffferlesnnynty。
Only keep the activity of bFGF and its receptors bind, and lacked the activity that stimulates further signal conduction, can not stimulate target cell propagation, itself and cationic-liposome have been hatched, be prepared into target liposomes.
Second technical problem to be solved by this invention provided the application of above-mentioned target liposomes carrier in pharmaceutical compositions.
The 3rd technical problem to be solved by this invention provided a kind of target liposomes complex.This cation lipid nanocrystal composition is to be encapsulated layer with above-mentioned target liposomes.
Further, be encapsulated with genophore or micromolecule antitumor drug in the encapsulated layer of above-mentioned target liposomes complex.
Wherein, the said gene carrier is an adenovirus vector, at least a in gland relevant viral vector, plasmid vector or the phage vector.
Wherein, above-mentioned adenovirus vector is a replication-defective adenoviral vector.
Wherein, be loaded with and can express the gene order of the antitumor cell factor on the said gene carrier.
Wherein, above-mentioned micromolecule antitumor drug is at least a in paclitaxel, amycin or the camptothecine.
The 4th technical problem to be solved by this invention provided the method for preparing of above-mentioned liposome.This method may further comprise the steps:
A, take by weighing DOTAP, Chol places in right amount to revolve and steams bottle, adds chloroform and makes it to dissolve fully
B, rotary evaporation in vacuo get liposome membrane, drying;
C, in liposome membrane, add the ultrasonic aquation of glucose solution and get blank liposome solutions;
D, the bFGF polypeptide is slowly joined in the blank liposome solution, slight concussion, hatching is spent the night, and obtains the targeting blank liposome.
The 5th technical problem to be solved by this invention provided the method for preparing of above-mentioned target liposomes complex.
When preparing the target liposomes complex of genophore, may further comprise the steps:
A, take by weighing DOTAP, Chol places to revolve and steams bottle, adds chloroform and makes it to dissolve fully;
B, rotary evaporation in vacuo get liposome membrane, drying;
C, in liposome membrane, add the ultrasonic aquation of 5% glucose solution and get blank liposome solutions;
D, tbFGF is slowly joined in the blank liposome solution, slight concussion, hatching is spent the night, and obtains the targeting blank liposome, genophore such as plasmid vector or viral vector and target liposomes is at room temperature hatched 30 minutes again, promptly gets.
When preparing the target liposomes complex of micromolecule antitumor drug, this method may further comprise the steps:
A, take by weighing DOTAP, Chol places to revolve and steams bottle, adds chloroform and makes it to dissolve fully;
B, rotary evaporation in vacuo get liposome membrane, drying;
C, in liposome membrane, add the ultrasonic aquation of 5% glucose solution and get blank liposome solutions;
D, employing thin-film ultrasonic method or freeze-thaw method are wrapped in the micromolecule antitumor drug in the blank liposome earlier, tbFGF are slowly joined in the liposome solutions again, slight concussion, and incubated overnight promptly gets.
Wherein, said method also comprises step e: the liposome after will hatching is pressed through 0.45 μ m polycarbonate membrane 4～8 times.
The 6th technical problem to be solved by this invention provided the application of above-mentioned liposome complex in being prepared in antineoplastic pharmaceutical compositions.
The 7th technical problem to be solved by this invention provides a kind of targeting antineoplastic medicine compositions, and it adds pharmaceutically above-mentioned liposome complex that the complementary composition of acceptable is prepared from.
According to a first aspect of the invention, for the stable FGFR target liposomes of the particle diameter that builds, investigated multiple liposome prescription.In order to obtain better effect, medicine fat ratio is screened, and the amount that the tbFGF polypeptide adds is estimated, obtained particle diameter and the stable target liposomes prescription of Zeta potential among the present invention.When being used to seal macromolecular drugs such as gene or genophore, the addition of tbFGF polypeptide is higher, as by weight: tbFGF polypeptide/TL molecule is that 20%～80%, 50%～70% left and right sides is preferable).When being used to seal the micromolecule antitumor drug, can only use lower tbFGF polypeptide, as by weight: tbFGF polypeptide/TL molecule be 2%～80% all can, but 2～10% left and right sides are preferable, amycin preferred 4%).
According to another aspect of the present invention, the method for preparing FGFR target liposomes complex of the present invention is confirmed and optimized.Preparing according to common technological parameter on the basis of cationic-liposome, adopting and hatch the compound tbFGF polypeptide of method.Also each technological parameters such as extruding number of times, incubation temperature are optimized in addition, also obtained the cationic-liposome of particle diameter simultaneously less than 200nm with the envelop rate that improves its parcel gene and small-molecule drug.
According to a further aspect of the invention, FGFR target liposomes of the present invention loads the Antioncogene carrier or the anti-tumor small molecular medicine can be used for tumor treatment, and can add pharmaceutically that the complementary composition of acceptable is prepared into antineoplastic pharmaceutical compositions again.
Beneficial effect of the present invention is: because bFGF and receptor thereof all have high-caliber expression on kinds of tumor cells and endothelial cells in tumor neogenetic blood vessels; TbFGF polypeptide and cationic-liposome are hatched in the present invention; Be prepared into the nanometer liposome that tbFGF modifies; It can carry out self assembly with DNA or viral vector and form targeted nano liposome/DNA complex, can wrap up the micromolecule antitumor drug again simultaneously.In addition; The present invention also obtains the bFGF peptide section (tbFGF) of truncate to the structure transformation of bFGF, comprise the 30-115 aminoacid of bFGF, has only kept the activity of bFGF and its receptors bind; And lacked the activity that stimulates the conduction of further signal; Can not stimulate target cell propagation, itself and cationic-liposome are hatched, be prepared into target liposomes.Transfection efficiency and stability after the experiment proof is used the stability of this target liposomes and sealed various antitumor drug all are improved simultaneously; Possesses body internal target tropism; The tumor tissues drug distribution is greatly improved, and clearance rate greatly reduces in the body of medicine simultaneously.The effect that can be well be transported to therapeutic gene and small-molecule drug specific tumor tissues selectively; And not only can wrap up the micromolecule antitumor drug but also can wrap up genomic medicine; Also can realize wrapping up micromolecule antitumor drug and genomic medicine simultaneously, be a kind of outstanding medicine-carried system that tumor is had the specific target tropism.Through carrying anti-tumor activity gene survivin; Reorganization survivin adenovirus, amycin, medicines such as paclitaxel show good anti-tumor effect in models such as kinds of tumors such as breast carcinoma, carcinoma of prostate, melanoma; Suppressed growth of tumor; And the life cycle of significant prolongation tumor-bearing mice, for the targeted therapy Products Development of tumor provides new thinking with using, have good clinical application prospect.
Description of drawings:
Fig. 1: the expression vector pET-32a (+) that the tbFGF polypeptide is adopted.
Fig. 2: the variation of particle diameter before and after the compound tbFGF polypeptide of empty liposome.
Fig. 3: the variation of Zeta potential before and after the compound tbFGF polypeptide of empty liposome.
Fig. 4: the tumor growth curve of FGFR target liposomes-pORF9-mSurvivinT34A complex treatment murine melanoma (B16).
Fig. 5: curve life cycle of FGFR target liposomes-pORF9-mSurvivinT34A complex treatment murine melanoma (B16).
Fig. 6: the tumor growth curve of FGFR target liposomes-amycin complex treatment mice prostate tumor (Tramp-C1).
Fig. 7: the survival curve of FGFR target liposomes-amycin complex treatment mice prostate tumor (Tramp-C1).
Fig. 8: the tumor growth curve of FGFR target liposomes-paclitaxel complex treatment murine melanoma (B16).
Fig. 9: the survival curve of FGFR target liposomes-paclitaxel complex treatment mice murine melanoma (B16).
Figure 10: the alginate experiment detects the effect that the FGFR target liposomes suppresses new vessels:
A. normal saline group, B. free paclitaxel group, C. common cation liposome paclitaxel group, D.FGFR target liposomes, E, dextran fluorescence intensity in the alginate ball in each group.
The specific embodiment
Below through to the description of specific embodiment of the invention explanation but do not limit the present invention.
The selection and the optimization of embodiment one liposome prescription
Material: 1, the trimethylammonio propane of 2-two oleoyls-3-(DOTAP) is purchased the company in Avanti; Cholesterol (Chol) is purchased the company in Sigma; TbFGF is carrier construction, expression preparation voluntarily, and purity is greater than 99%.
The construction and expression of tbFGF: analyst bFGF sequence; The primer of design amplification tbFGF (aa.30-115); Forward primer 5 ' end introduces restricted enzyme Kpn I restriction enzyme site and enterokinase (EK enzyme) is cut the site; Downstream primer 5 ' end is introduced restricted enzyme Hind III restriction enzyme site, and is synthetic by the handsome Bioisystech Co., Ltd in Shanghai.
Forward primer (Seq ID No.2):
5’GG
GGTACC 
AAGCGGCTGTACTGCAAAAACG 3’。
Downstream primer (Seq ID No 3):
5’CCC
AAGCTTAGTAAGTATTGTAGTTATTAGATTC 3’。
With pBLAST45-hbFGF (available from American I nvivogen company) is template amplification tbFGF fragment, with inserting expression vector pET-32a (+) (available from U.S. Novagen company, structural representation is seen Fig. 1) behind the PCR product KpnI/Hind III double digestion.After identifying correctly, with recombiant plasmid pET32a-tbFGF transformed into escherichia coli BL21 (DE3) competent cell, IPTG induces destination protein to express.The result shows that pET32a-tbFGF/BL21 (DE3) induces the fusion rotein that can express consistent with estimated molecular weight (about 21kDa) down at IPTG, and expression reaches more than 40%.Broken bacterium supernatant obtains purification through one step of metal chelating column affinity chromatograph, and fusion rotein Trx-tbFGF purity can reach more than 95%, and output can reach 400-500mg/L.
Through the fusion rotein Trx-tbFGF of Ni column purification, can obtain thioredoxin and molecular weight behind the EK enzyme enzyme action is the tbFGF polypeptide about 9.1kDa, and purity can reach more than 99%.
Through measuring, the sequence of this polypeptide is:
n’-krlycknggfflrihpdgrvdgvreksdphiklqlqaeergvvsikgvcanrylamkedgrllaskcvtdecffferlesnnynty-C’
In the activity that stimulates cellular proliferation of external use NIH-3T3 cell detection tbFGF, the result shows: the ED50 value of tbFGF is 500ng/ml, than the proteic ED50 value of standard bFGF (＜5ng/ml) high more than 100 times.Explain that this tbFGF polypeptide stimulates the active basic forfeiture of target cell, can be used for later targeting experimentation.
This tbFGF polypeptide of mass preparation is used for the experiments research of the specific embodiment of the invention.
Aspect material, find to use the liposome toxicity of DOTAP/Chol system gained very low through preliminary experiment, and more help compound tbFGF, so lipid molecular is selected DOTAP and Chol for use.
Through changing the ratio (mol ratio) of DOTAP: Chol, observe variation to tbFGF compound back particle diameter and surface charge.The ratio (mol ratio) of finding DOTAP: Chol is 1: 1 o'clock, can obtain stable particle diameter and electric charge, and the result sees Fig. 2.
On the ratio of compound tbFGF; When the ratio (mol ratio) that keeps DOTAP: Chol is 1: 1; The tbFGF coupling that adds different proportion; The variation of investigation particle diameter and surface potential on the tbFGF basis of different proportion is as DOTAP: Chol=1: 1 (mol ratio), can obtain stable particle diameter and surface charge when the tbFGF polypeptide accounts for 10～80%.When the tbFGF polypeptide accounts for 65% (weight ratio), its compound polypeptide accounts for 65% o'clock of whole liposome weight ratio can obtain stable particle diameter and surface charge, and the result sees Fig. 2.
The further optimization of embodiment two targeting blank liposome preparation process conditions
In the whole technical process; The positive charge of the compound main dependence cationic-liposome of blank liposome and tbFGF polypeptide and tbFGF polypeptide with negative charge between electrostatic force be achieved, can obtain the targeting blank liposome through hatching jointly of cationic-liposome and tbFGF polypeptide.In order to obtain stable targeting blank liposome, the compound condition of our tbFGF polypeptide and cationic-liposome is optimized.Wherein, number of freezing and thawing has considerable influence to the tbFGF envelop rate.
At first, investigate number of freezing and thawing to the bonded influence of tbFGF.The targeting blank liposome is with after different proportion tbFGF combines, and the envelop rate when multigelation 1,2,3,4,5,6 times changes sees table 1.Data show, beginning be during freeze thawing, and the increase of number of freezing and thawing helps combining of liposome and tbFGF; But after the multigelation 6 times, its particle diameter can become greatly, and envelop rate is not had obvious variation.Freeze thawing all can achieve the goal for 2-6 time basically, and best number of times is 3 times.
Table 1: number of freezing and thawing is to the influence of tbFGF recombination rate:
Secondly, we are optimized the freeze thawing incubation condition.On incubation condition is investigated, investigate the influence of incubation temperature, brooding time respectively to envelop rate.Select 4 ℃, 8 ℃, 10 ℃ on the incubation temperature, select 40,60,80,100,120 minutes on the brooding time.The result shows that incubation temperature is little to the envelop rate influence, should keep water-bath below 10 ℃, 4 ℃ of the bests; On the brooding time, 40～80 minutes obtain on the high envelop rate satisfied, 60 minutes the bests.
Cross the investigation of film extruding degerming: liposome is through after the freeze thawing, and obtaining is that the particle size distribution and the mean diameter of a broad is also bigger than normal, if can obtain narrower particle size distribution and improve mean diameter through film extrusion, improves overall performance.Can also reach the purpose of degerming simultaneously through the extruding of 0.2mm film.
Through above-mentioned process optimization; Can obtain the blank liposome preparation technology of a best targeting FGFR: accurately take by weighing DOTAP, Chol by prescription, place eggplant-shape bottle, add chloroform: the organic solvent dissolution of methanol=1: 1; 33 ℃ of rotary evaporation in vacuo get liposome membrane; Put into vacuum drying oven room temperature drying under vacuum overnight, add the ultrasonic aquation of ultra-pure water (200W, 10 minutes) and get blank liposome solutions.With tbFGF (by weight: tbFGF polypeptide/TL molecule can be 20%～80%; Be good about 65%) add liposome solutions, 4 degrees centigrade of hatchings are spent the night, and re-use squeezing and pressing method through the degerming of 0.20mm polycarbonate membrane; Get tbFGF liposome complex, i.e. targeting blank liposome.The suitable complex with preparation and gene or genophore of this targeting blank liposome.
The preparation technology of embodiment three, target liposomes parcel DNA or adenovirus
The preparation of target liposomes gene composite then is to hatch through targeting blank liposome and DNA or the suitable ratio of adenovirus, and self assembly forms.
The targeting blank liposome that embodiment two obtains and DNA in 1: 3 ratio (weight ratio) mix, 4 ℃ of incubated overnight can obtain target liposomes plasmid dna complex compound.
Equally, the targeting blank liposome that obtains of embodiment two and recombinant adenovirus are in proportion: 2.5 * 10
9VP recombinant adenovirus/mg cationic-liposome mixes, and incubated at room 10～30 minutes can obtain the target liposomes adenovirus composition.
Embodiment four, target liposomes parcel micromolecule preparing anti-tumor medicine technology
Parcel micromolecule preparing anti-tumor medicine technology is example with the amycin.Its preferable preparation technique is following:
The scheme of reference implementation example two; Accurately take by weighing a certain proportion of DOTAP, Chol, amycin by prescription and place eggplant-shape bottle; Add chloroform: the organic solvent dissolution of methanol=1: 1,33 ℃ of rotary evaporation in vacuo get liposome membrane, put into vacuum drying oven room temperature drying under vacuum overnight; Add the ultrasonic aquation of ultra-pure water (200W, 10 minutes) and get Evacet solution.Again with tbFGF add liposome solutions (by weight: but the scope that the tbFGF polypeptide/TL molecule dish is used is bigger; 4% left and right sides is preferable); The four degrees celsius hatching is spent the night, and re-uses squeezing and pressing method through the 0.45mm polycarbonate membrane, obtains target liposomes amycin complex.Equally also obtain the complex of target liposomes envelope paclitaxel of the present invention.
Embodiment five FGFR target liposomes parcel pCMV-GFP measures transfection efficiency in vitro
In order to verify the targeting property after the FGFR target liposomes wraps up gene; We carry out external checking with the cell strain of several kinds of high expressed FGFR and cell strain low or that do not express FGFR, detect the transfection efficiency of FGFR target liposomes parcel pCMV-GFP in these several kinds of cell strains.
The cell strain of high expressed FGFR: human hepatoma cell strain Hep-G2, mice prostate gland cancer cell strain Tramp-c1, human oophoroma cell line A2780, Human umbilical vein endothelial cells strain HUVEC.
Low or do not express the cell strain of FGFR: mouse colonic cell strain CT26, mouse ovarian JEG-3 4T1.
Plasmid: pCMV-GFP (available from U.S. Clontech company).
Operating procedure: cell inoculation is (40000 cells/well) in six orifice plates, treat that every porocyte covered with at 75% o'clock, the beginning transfection experiment.The cationic-liposome that 10 μ g tbFGF are modified slowly joins 2 μ g pCMV-GFP (all diluting with serum-free medium); After hatching 20～40 minutes under the room temperature; Join in six orifice plates, after 4 hours, all changed the serum-free medium in each hole into blood serum medium; Observing under the fluorescence microscope after 24 hours has green fluorescence to occur in the cell, harvesting detects the fluorescence intensity of respectively organizing cell with flow cytometer.
The operation by specification of reference standard cationic-liposome Lipofectamine 2000 (available from American I nvitrogene company) carries out.
Experimental result is seen table 2, and the result shows that FGFR target liposomes of the present invention can improve the transfection efficiency of FGFR high expressing cell strain specifically, and does not influence the low or transfection efficiency of expression cell line not of FGFR, has FGFR targeting property.
The transfection efficiency of liposome in different cell strains that table 2 is dissimilar
Below through the mode of Test Example beneficial effect of the present invention is done further detailed description.
Test Example one, FGFR target liposomes parcel plasmid pORF9-mSurvivinT34A treatment murine melanoma
Suppress the activity in vivo that the growth of tumor situation shows target liposomes complex of the present invention through measuring.
Animal: 8 the week age C57 mice, every the inoculation 2 * 10
5Divide into groups behind the B16 cell (10 every group), treat to lay one's hand on and begin the tail intravenously administrable during tumor, weekly twice, successive administration eight times was measured gross tumor volume in per three days.Investigate tumor growth and generation phase, after waiting to stop to treat, get each tissue pathological slice analysis.
Plasmid: pORF9-mSurvivinT34A (available from American I nvivogen company)
Divide into groups: FGFR target liposomes-pORF9-mSurvivinT34A group (25 μ g plasmids/only/inferior)
FGFR cationic-liposome-zero load group (pORF9) (25 μ g plasmids/only/inferior)
Common cation liposome-survivin group pORF9-mSurvivinT34A group (25 μ g plasmids/only/inferior)
Common cation liposome-zero load group (pORF9) (25 μ g plasmids/only/inferior)
The normal saline group
Press down the tumor curve and see Fig. 4; The result shows that it is 40.3% that tbFGF target liposomes of the present invention-pORF9-mSurvivinT34A complex treatment mouse melanin suppresses the tumor growth rate; P＜0.001; And the inhibition tumor growth rate of FGFR target liposomes-pORF9-mSurvivinT34A complex treatment mouse melanin is 68.3%, and p＜0.001 has significant difference.
Life cycle, curve was seen Fig. 5, and the result shows the life cycle of FGFR target liposomes-pORF9-mSurvivinT34A complex ability significant prolongation tumor-bearing mice.
The Antitumor Effects of Test Example two, tbFGF target liposomes parcel anticancer drugs, doxorubicin
Divide into groups: tbFGF target liposomes parcel amycin group (the amycin dosage of parcel is 100 μ g/)
Conventional liposome parcel amycin (the amycin dosage of parcel is 100 μ g/)
Empty liposome group (liposome dosage is 500 μ g/)
Free amycin group (amycin dosage is 100 μ g/)
The normal saline group
Animal: 8 age in week the C57 mice, divide into groups behind the inoculation mice prostate gland cancer cell strain Tramp-C1 cell (10 every group), treat to lay one's hand on and begin the tail intravenously administrable during tumor, weekly twice, successive administration eight times was measured the tumor body in per three days.Investigate tumor growth and generation phase, after waiting to stop to treat, get each tissue pathological slice analysis.
Press down the tumor curve and see Fig. 6; The result shows: the tumor control rate of free amycin is merely 68.2%; And common cation liposome amycin tumor control rate does not improve (51.7%); And FGFR target liposomes parcel amycin treatment carcinoma of prostate, its tumor control rate is 87.9%, p＜0.001.
Survival curve is seen Fig. 7, and the result shows: after the FGFR target liposomes parcel amycin treatment carcinoma of prostate, the production rate of mice also had the survival of 30% mouse later on up to 90 days, and other several groups of mouse are all dead at 78 days.The life cycle of FGFR target liposomes parcel amycin ability significant prolongation tumor-bearing mice.
The Antitumor Effects of Test Example three, FGFR target liposomes parcel anti-cancer medicine paclitaxel
Divide into groups: FGFR target liposomes parcel paclitaxel group (dose of paclitaxel of parcel is 200 μ g/)
Conventional liposome parcel paclitaxel (dose of paclitaxel of parcel is 200 μ g/)
Empty liposome group (liposome dosage is 800 μ g/)
Free paclitaxel group (dose of paclitaxel of parcel is 200 μ g/)
The normal saline group
Animal: 8 age in week the C57 mice, divide into groups behind the inoculation mouse melanin tumor cell B16 (10 every group), treat to lay one's hand on and begin the tail intravenously administrable during tumor, weekly twice, successive administration eight times was measured the tumor body in per three days.Investigate tumor growth and generation phase, after waiting to stop to treat, get each tissue pathological slice analysis.
Press down the tumor curve and see Fig. 8; The result shows: free paclitaxel is merely 35.7% to the tumor control rate of murine melanoma; The tumor control rate of common cation liposome paclitaxel increases (49.9%, p＜0.001), and FGFR target liposomes parcel paclitaxel treatment murine melanoma obtains good restraining tumor growth effect; Its tumor control rate reaches 77.7%, p＜0.001.
Survival curve is seen Fig. 9; FGFR target liposomes parcel paclitaxel treatment murine melanoma; Behind the common cation liposome paclitaxel; Mice is all dead at 36 days, and FGFR target liposomes parcel paclitaxel group also has the survival of 50% mouse, and the survival of 30% mouse is arranged up to 60 days of off-test.Experimental result shows the life cycle of this target liposomes ability significant prolongation tumor-bearing mice.
More than three Test Example show that FGFR target liposomes complex of the present invention can effectively suppress growth of tumor, prolong the life cycle of tumor-bearing mice, have significant antitumous effect.
Test Example four, alginate experiment proof FGFR target liposomes suppress the effect of tumor-blood-vessel growth
Mouse colonic cell CT26 is resuspended in the normal saline solution of 1.6% alginate, again it slowly joined in the 250mmol/L calcium chloride solution, form alginate granule (1 * 10
5Cell/granule).Four alginate granules of every subcutaneous implantation of BalB/c mouse back afterwards, the operation stitching wound.Since second day, be administered once in per three days, and in the 12nd day; Every mouse tail vein injection 0.1mL FITC-dextran solution (100mg/kg); Take out the alginate granule after 20 minutes, take pictures, and detect the content of FITC-glucosan in each granule with spectrofluorophotometer.
Experimental result is seen Figure 10; Alginate granule behind the cationic-liposome parcel paclitaxel treatment that bFGF modifies in tumor tissues does not almost observe tumor vessel, and in common cation liposome paclitaxel treatment group and free paclitaxel group, still can observe tumor vessel.And a large amount of tumor vessels exists in the normal saline group.Experimental result shows that the FGFR target liposomes has tumor vascular targeting property.
The above more excellent specific embodiment is that the present invention is further illustrated; But be not limitation of the scope of the invention; Those skilled in the art are according to basic thought of the present invention; Can make various modification or improvement, only otherwise break away from basic thought of the present invention, all within the scope that inventive concept of the present invention and claims of being enclosed define.
SEQUENCE LISTING
< 110>Sichuan University
< 120>the receptor-mediated blood-vessels target liposome vectors of fibroblast growth factor and method for preparing and purposes
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Claims (16)
1. target liposomes; It is characterized in that: the composition following proportioning is prepared from: 1; The trimethylammonio propane of 2-two oleoyls-3-, cholesterol and tbFGF polypeptide, its proportioning is: 1, the mol ratio of trimethylammonio propane of 2-two oleoyls-3-and cholesterol is 1; The trimethylammonio propane of 2-two oleoyls-3-: cholesterol=2: 1～2; The weight content of tbFGF polypeptide is 1, and 2～80% of trimethylammonio propane of 2-two oleoyls-3-and cholesterol gross weight, said tbFGF amino acid sequence of polypeptide are shown in the SEQ ID NO.1.
2. target liposomes according to claim 1, it is characterized in that being prepared from the composition of following proportioning: the weight content of said tbFGF polypeptide is 1,3～70% of trimethylammonio propane of 2-two oleoyls-3-and cholesterol gross weight.
3. each described target liposomes application in pharmaceutical compositions of claim 1～2.
4. target liposomes complex, it is characterized in that: with the described target liposomes of claim 1 is encapsulated layer.
5. target liposomes complex according to claim 4 is characterized in that being encapsulated with in the described encapsulated layer expression vector or micromolecule antitumor drug.
6. target liposomes complex according to claim 5 is characterized in that described expression vector is at least a in adenovirus vector, gland relevant viral vector, plasmid vector or the phage vector.
7. target liposomes complex according to claim 6 is characterized in that described adenovirus vector is a replication-defective adenoviral vector.
8. target liposomes complex according to claim 7 is characterized in that being loaded with and expressing on the described expression vector gene order of the antitumor cell factor.
9. target liposomes complex according to claim 5 is characterized in that described micromolecule antitumor drug is at least a in paclitaxel, amycin or the camptothecine.
10. prepare the method for each described target liposomes of claim 1～2, it is characterized in that: may further comprise the steps:
A, take by weighing 1, the trimethylammonio propane of 2-two oleoyls-3-, cholesterol place in right amount to revolve and steam bottle, add chloroform and make it to dissolve fully;
B, rotary evaporation in vacuo get liposome membrane, drying;
C, in liposome membrane, add the ultrasonic aquation of glucose solution and get blank liposome solutions;
D, the tbFGF polypeptide is slowly joined in the blank liposome solution, slight concussion, hatching is spent the night, and obtains the targeting blank liposome.
11. a method for preparing each described target liposomes complex of claim 4～9 is characterized in that may further comprise the steps:
A, take by weighing 1, the trimethylammonio propane of 2-two oleoyls-3-, cholesterol place to revolve and steam bottle, add chloroform and make it to dissolve fully;
B, rotary evaporation in vacuo get liposome membrane, drying;
C, in liposome membrane, add the ultrasonic aquation of 5% glucose solution and get blank liposome solutions;
D, tbFGF is slowly joined in the blank liposome solution, slight concussion, hatching is spent the night, and obtains the targeting blank liposome, expression vector and target liposomes is at room temperature hatched 10～30 minutes again, promptly gets.
12. a method for preparing each described target liposomes complex of claim 4～9 is characterized in that may further comprise the steps:
A, take by weighing 1, the trimethylammonio propane of 2-two oleoyls-3-, cholesterol place to revolve and steam bottle, add chloroform and make it to dissolve fully;
B, rotary evaporation in vacuo get liposome membrane, drying;
C, in liposome membrane, add the ultrasonic aquation of 5% glucose solution and get blank liposome solutions;
D, employing thin-film ultrasonic method or freeze-thaw method are wrapped in the micromolecule antitumor drug in the blank liposome earlier, tbFGF are slowly joined in the liposome solutions again, slight concussion, and incubated overnight promptly gets.
13. the method for preparing the target liposomes complex according to claim 11 is characterized in that: also comprise step e: the liposome after will hatching is pressed through 0.45 μ m polycarbonate membrane 4～8 times.
14. the method for preparing the target liposomes complex according to claim 12 is stated is characterized in that: also comprise step e: the liposome after will hatching is pressed through 0.45 μ m polycarbonate membrane 4～8 times.
15. each described target liposomes complex application in being prepared in antineoplastic pharmaceutical compositions of claim 4～9.
16. a targeting antineoplastic medicine compositions, adding pharmaceutically each described target liposomes complex of claim 4～9, the complementary composition of acceptable is prepared from.
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| CN102114000B (en) * | 2009-12-31 | 2013-08-21 | 复旦大学 | Co-feeding lipid nano-delivery system for medicine carrying |
| CN106265518B (en) * | 2016-08-31 | 2018-12-07 | 河南佰柯生物科技有限公司 | FGF liposome and preparation method thereof |
| CN107019673A (en) * | 2017-04-13 | 2017-08-08 | 四川省人民医院 | A kind of Paclitaxel liposome preparation with tumor-targeting function and its preparation method and application |
| CN107041871B (en) * | 2017-04-24 | 2020-12-29 | 四川省人民医院 | Cationic liposome of kinase inhibitor, composition of cationic liposome and siRNA and preparation method of cationic liposome |
| CN110575549B (en) * | 2018-06-08 | 2021-11-05 | 四川大学华西医院 | tbFGF ligand-modified liposome with tumor active targeting function and preparation method and application thereof |
| CN109125741B (en) * | 2018-08-13 | 2022-02-11 | 四川大学 | Self-assembled ternary complex preparation of hyaluronic acid/DOTAP/survivin coding gene and preparation method thereof |
| CN113384712A (en) * | 2021-06-11 | 2021-09-14 | 潍坊医学院 | Co-loaded liposome based on simultaneous killing of tumor cells and CAFs and preparation method thereof |
| CN114652820B (en) * | 2022-03-22 | 2024-02-06 | 中国药科大学 | Cationic liposome nanoparticle and preparation method and application thereof |
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