CN106822920A - The PEDF gene composites of tumour cell folacin receptor targeting - Google Patents
The PEDF gene composites of tumour cell folacin receptor targeting Download PDFInfo
- Publication number
- CN106822920A CN106822920A CN201610668056.3A CN201610668056A CN106822920A CN 106822920 A CN106822920 A CN 106822920A CN 201610668056 A CN201610668056 A CN 201610668056A CN 106822920 A CN106822920 A CN 106822920A
- Authority
- CN
- China
- Prior art keywords
- pedf
- folic acid
- modified
- plasmid
- liposome
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
- A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
Abstract
The present invention relates to field of medicaments, and in particular to a kind of PEDF gene composites of tumour cell folacin receptor targeting and its production and use.The present invention constructs a kind of PEDF gene composites of modified with folic acid, the compound can utilize the folacin receptor of tumor locus expression high that the plasmid for encoding PEDF is selectively delivered into tumour cell, induce its apoptosis, suppress tumor cell proliferation, invasion and attack and transfer, and then antitumor action is played, reach preferable antitumous effect.
Description
Technical field
The present invention relates to field of medicaments, and in particular to a kind of PEDF gene composites of tumour cell folacin receptor targeting and
Preparation Method And The Use.
Background technology
Pigment epidermal derived factors (PEDF) are a secreting type sugar eggs for the endogenic 50kDa for spreading all over body tissue
In vain, it was found first and reported as the factor in retinal endothelial cell in 1989.Development over time, PEDF's
MRNA is proved to be deposited extensively in most fetuses or adult normal histocyte (such as blood plasma, heart, lungs, ovary) and tumour
.PEDF has directly suppression cancer characteristic, is mainly realized by antitumor apoptosis, antitumor differentiation and antitumor propagation；Together
When also have indirectly suppression cancer characteristic, mainly by the apoptosis of VEGF (VEGF) and inducing endothelial cell so that
Anti-tumor neovascularization.PEDF has a good therapeutic effect as anticancer factor in a series of tumor researches, such as lung cancer,
Liver cancer, breast cancer, prostate cancer, oophoroma, melanoma, osteosarcoma, colon cancer and cervical carcinoma etc..
The PEDF of research is now widely used in mainly to be obtained from mammalian cell such as the cells of HEK 293, it is expensive,
And consumption is up to milligram level in vivo, is clinically difficult to large-scale application.Directly treated with PEDF genes, with dosage
Small advantage, is mode of administration of the current PEDF for the great prospect of clinical therapy of tumor.It is presently used for transmitting PEDF genes
What carrier was reported has retrovirus, adenovirus, slow virus, plasmid, cationic-liposome, chitosan microball etc..Reverse transcription disease
There is the technological deficiencies such as immunogenicity, potential infectious and targeting difference in poison, adenovirus and slow virus this several viral vectors.
The characteristics of plasmid has easy, safe, inexpensive, and the DNA of larger dose can be carried, but because nucleic acid in vivo enzyme etc. is to naked
The degradation of DNA, the time of gene expression is very short, it is impossible to clinical practice；Cationic-liposome is at present using relatively broad
Gene transfer vector, there is channel genes efficiency high；Shitosan is also a kind of gene with potential using value
Transfection carrier, but the carrier of load PEDF genes of report is chitosan microball at present, there is that particle diameter is larger, cellular uptake efficiency
Low technological deficiency.
In the mandate patent of invention 201010136539.1 of this seminar early stage, disclose and delivered with PLGA nanoparticles
The technology of PEDF genes, the nanoparticle can realize the high-efficiency delivery of PEDF genes in vivo；But tumour cell is lacked and is selected
Property, need further raising in the concentration of tumor locus and gene expression dose.Therefore, the present invention intends utilizing tumor cell surface
The acceptor of specifically expressing this molecular biological characteristics, design the PEDF gene delivery vectors of active targeting.
Folacin receptor (folate receptor, FR) is a kind of glycosylated membrane glycoprotein, is generally had in the normal tissue
The expression of reduced levels, but in many malignant tumours, there is high level expression in FR.Document report, various epithelial origins it is swollen
Knurl includes oophoroma, kidney, prostate cancer, breast cancer, stomach cancer, cervical carcinoma, the cancer of the brain, lung cancer, incidence cancer and colorectal cancer
Deng the expression high for having folacin receptor.Using can the expression high of specially recognizing tumor cells surface folacin receptor small molecule folic acid
Modification PEDF gene delivery vectors, it is possible to achieve the targeted delivery of tumor-selective, further improve the antitumor of PEDF genes
Effect.
Inventor intends building a kind of PEDF gene composites of modified with folic acid, using the folacin receptor of tumor locus expression high
The plasmid for encoding PEDF is selectively delivered to tumour cell；The PEDF of tumour cell high efficient expression, by suppressing tumour cell
Propagation, inducing apoptosis of tumour cell, induced tumor cell differentiation are ripe, suppress the number of mechanisms hair such as tumor cell invasion and transfer
Antitumor action is waved, preferable antitumous effect is reached.
The content of the invention
Technical problem solved by the invention is to provide a kind of PEDF gene composites of modified with folic acid, it is therefore intended that targeting
Delivering PEDF genes, using the PEDF of high efficient expression, play preferable antitumor action to tumour cell.
Said composition contains：Express plasmid, the modified with folic acid liposome of PEDF.Can add other it is pharmaceutically acceptable into
Point, also can be without other compositions.Wherein, the plasmid of expression PEDF and the mass ratio of modified with folic acid liposome are 1:1~1:15,
Preferably 1:2~1:15.
The plasmid of the expression PEDF in the pharmaceutical composition, plasmid can be the plasmid that can arbitrarily obtain, such as laboratory
The plasmid of structure, or the plasmid of commercialization, such as pVITRO2, pAAV2, pGenesil2.1, pGenesil2.4, pCMV-
Script, pBlast49 etc..
Modified with folic acid liposome in said composition, containing matrix material and modified with folic acid lipid, can add other pharmacy
Upper acceptable composition, also can be without other compositions；Wherein, folic acid accounts for the 0.01~5% of TL molal quantity, preferably
0.05~2.5%.
Matrix material used is including in phosphatide and its derivative or cholesterol and its derivative in modified with folic acid liposome
One or more；Modified with folic acid lipid used is coupled with matrix material by folic acid and is formed, and centre can use interval base,
Can be without spacer group, spacer group can select any energy and folic acid be coupled into the base being connected with matrix material by chemical reaction
Group, such as conventional polyglycol chain (PEG, the polyethylene glycol of 200~10000Da of molecular weight), aminocaproic acid etc..
Specifically, as matrix material phosphatide and cholesterol species is a lot, can be usually used in preparing fat using formulation art
The phosphatide and cholesterol type of plastid.Specifically, when preparing the liposome of the dual-gene compositions of PEDF of modified with folic acid, use
Matrix material is neutral lipid, negative charged lipid or lipid material, including：Soybean lecithin, lecithin, cephalin, sheath
Phosphatide (SM), phosphatid ylcholine (PC), phosphatidyl-ethanolamine (PE), DPPC (DPPC), DSPC
(DSPC), dimyristoyl phosphatidyl choline (DMPC), phosphatidic acid (PA), phosphatidyl glycerol (PG), phosphatidylinositols (PI), phosphorus
Acyl serine (PS), double spermaceti phosphatidic acids (DCP), stearmide (stearylamine, SA), N- [1- (2,3- dioleoyls
Base) propyl group -]-N, N, N- trimethyl ammonium chloride [DOTMA], N- [1- (2,3- dioleoyl) propyl group]-N- (2- (arginine base acyls
Amine) ethyl-N, N-dimethylammonium teifluoroacetate, DOSPA), GERBU Adjuvant 100
(DDAB), 1,2- dioleoyls -3- phosphocholines (DOPC), 1,2-dioleoyl-3-trimethyl ammonium propane (chloride salt)
(DOTAP), 1,2-octacosyl-SN- glycerol-3-phosphates monoethanolamine (DSPE), cholesterol, [N- (N', N'- dimethylaminos
Ethane)-carbamoyl] cholesteric alcohol hydrochloride (DC-Chol), and other derivatives of phosphatide or cholesterol other derivatives
Thing, including polyglycol derivatization matrix material, etc..
The modified with folic acid liposome that the present invention is provided, specific preparation method can use the preparation method of conventional liposome, including
Various preparation methods such as membrane process, reverse evaporation, injection method, ultrasonic method, freeze-thaw method.
Brief description of the drawings
The blank liposome (FLP) of Fig. 1 modified with folic acid and the PEDF gene composites (FLP/PEDF) of modified with folic acid it is saturating
Penetrate electromicroscopic photograph.
The PEDF gene composites (FLP/PEDF) of Fig. 2 modified with folic acid can substantially reduce tumour, improve Tumor growth inhibition
Rate.
The PEDF gene composites (FLP/PEDF) of Fig. 3 modified with folic acid can substantially reduce ascites generation.
The PEDF gene composites (FLP/PEDF) of Fig. 4 modified with folic acid can substantially reduce tumor nodule quantity.
Specific embodiment
Illustrated below by way of specific description of embodiments of the present invention but do not limit the present invention.
It is prepared by the modified with folic acid lipid that embodiment 1~5 is not added with interval base
Specifically feed intake as follows：
Concrete operations are：By folic acid (0.42mmol), NHS (0.5mmol), EDCI (0.5mmol) and triethylamine (4mmol)
It is dissolved in 5ml anhydrous dimethyl sulfoxides, the DMSO for dropping to phosphatide containing amino or derivatives thereof (DSPE, SA, 0.5mmol) is molten
Liquid.After 25 DEG C~30 DEG C reaction about 120h~144h, reaction solution is transferred to bag filter (MWCO=1000Da), respectively with 20%
(v/v) DMSO and water are dialysis medium dialysis, after dialysis 14 days, in transfer dialysis sample to silk mouthful bottle, are freezed, obtain final product folic acid-
Cholesterol or folic acid-phosphatide (i.e. modified with folic acid lipid).Folic acid reaction, purifying and lyophilized whole process lucifuge, product keep in dark place in
In drier.
It is prepared by modified with folic acid lipid of the embodiment 6~10 with α-carboxyl-omega-amino polyethylene glycol as interval base
Specifically feed intake as follows：
| Embodiment is numbered | 6 | 7 | 8 | 9 | 10 |
| α-carboxyl-omega-amino molecular weight polyethylene glycol (dalton) | - | 200 | 1000 | 3500 | 5000 |
| Aminocaproic acid | + | - | - | - | - |
| Cholesterol | + | + | - | - | - |
| Hydroxyl modified DC-Chol | - | - | + | - | - |
| PG | - | - | - | + | - |
| DCP | - | - | - | - | + |
Concrete operations are：Take α-carboxyl-omega-amino polyethylene glycol (amino terminal protection) or aminocaproic acid (amino terminal
Protection) 0.5mmol, matrix material (cholesterol or phosphatide) 0.75mmol, DMAP 0.75mmol and 1- ethyl-3-
(3- dimethylaminopropyls)-carbodiimides 0.75mmol is dissolved in 100ml dichloromethane, reacts about 72~96h.Decompression rotation
Organic solvent is evaporated off, amino-polyethyleneglycols cholesterol crude product is vacuum dried to obtain.Weigh folic acid 1mmol, N- hydroxysuccinimidyl acyl
Imines 2.5mmol, 1- ethyl-3-(3- dimethylaminopropyls)-carbodiimides 2.5mmol and triethylamine 10mmol is dissolved in
In the anhydrous DMSO of 50ml, about 0.5mmol amino-polyethyleneglycols cholesterol crude products are added, 25~30 DEG C are reacted about 72~96h, instead
Answer liquid to be transferred to bag filter (MWCO=3500Da), be respectively dialysis medium dialysis with 20% (v/v) DMSO and water, after dialysing 7 days,
In transfer dialysis sample to silk mouthful bottle, freeze, obtain final product FA-PEG-Chol or folic acid-PEG-DSPE (i.e. leaf
Acid modification lipid).Folic acid reaction, purifying and lyophilized whole process lucifuge, product keep in dark place in drier.
It is prepared by modified with folic acid lipid of the embodiment 11~15 with double amino-polyethyleneglycols as interval base
Specifically feed intake as follows：
| Embodiment is numbered | 11 | 12 | 13 | 14 | 15 |
| Double amino-polyethyleneglycols molecular weight (dalton) | 400 | 2000 | 5000 | 1000 | 1500 |
| Cholesterol | + | - | + | - | - |
| Hydroxyl modified DOPC | - | + | - | + | + |
Concrete operations are：By lipid (cholesterol or phosphatide) (10mmol), succinic anhydride (50mmol), DMAP (5mmol)
It is dissolved in 50mL dichloromethane, 45 DEG C of vigorous reflux, stirs 48h, obtains succinylated lipid (cholesterol or phosphatide).Take succinyl
Change lipid (4mmol), 1- ethyls-3-(3- dimethylaminopropyls)-carbodiimides (10mmol), N-hydroxy-succinamide
(10mmol) is dissolved in a small amount of dichloromethane, in dropping to double amino-polyethyleneglycols (5mmol) dichloromethane solution of 100ml,
Reaction about 72h~96h is stirred at room temperature.Reaction solution is done with 60 mesh~80 mesh silica gel mixed samples, 300 mesh~400 mesh silica gel wet method dress posts
Method loading.With methylene chloride-methanol system as eluant, eluent, ratio is from 80:1,60:Isosorbide-5-Nitrae 0:1 to 20:1 (volume ratio, v/v), receives
Eluent of the collection containing target product, merges concentration and removes organic solvent, obtains amino-polyethyleneglycols-cholesterol.By folic acid
(0.42mmol), NHS (0.5mmol), EDCI (0.5mmol) and triethylamine (4mmol) are dissolved in 5ml anhydrous dimethyl sulfoxides, drop
The DMSO solution of ammonification base PEG-CHOL (0.21mmol).After 25 DEG C~30 DEG C reaction about 120h~144h, will react
Liquid is transferred to bag filter (MWCO=1000Da), is respectively dialysis medium dialysis with 20% (v/v) DMSO and water, after dialysing 14 days,
In transfer dialysis sample to silk mouthful bottle, freeze, obtain final product FA-PEG-Chol or folic acid-PEG-DSPE (i.e. leaf
Acid modification lipid).Folic acid reaction, purifying and lyophilized whole process lucifuge, product keep in dark place in drier.
The preparation of the PEDF gene composites of the modified with folic acid of embodiment 16~21
Modified with folic acid lipid is taken, is first dissolved with 5% glucose solution, the micella that the modified with folic acid lipid of 1mg/ml is obtained is molten
Liquid.Take 20mg matrix materials again, add chloroform to make dissolving, 37 DEG C of chloroform removed under pressure, the time is 1h, then in room temperature, Gao Zhen
More than 6h, glucose solution aquation demoulding, 60 DEG C of bath temperature, time 1h are kept under empty condition；Suspension after demoulding is transferred to
In cillin bottle, water bath sonicator while hot, 60 DEG C of bath temperature, ultrasonic 15min, 0.22 μm of filtering with microporous membrane, filtrate and PEDF bases
Because mixing in mass ratio, 30min is incubated at room temperature, is designated as P-LP/PEDF.Take the micellar solution and P-LP/ of modified with folic acid lipid
PEDF presses upper table mixed in molar ratio, is placed in 37 DEG C of constant temperature air bath oscillators, after 100rpm shakings 1h, obtains final product modified with folic acid
PEDF gene composites, be designated as F-P-LP/PEDF.
It is prepared by the PEDF gene composites of the modified with folic acid of embodiment 22~27
Matrix material is taken in eggplant type flask, chloroform-methanol mixed solvent dissolves, and 37 DEG C are removed under reduced pressure organic solvent, when
Between be 1h, then under room temperature, high vacuum condition keep more than 6h, glucose solution aquation demoulding, 60 DEG C of bath temperature, time
1h；Suspension after demoulding is transferred in cillin bottle, while hot water bath sonicator, 60 DEG C of bath temperature, ultrasonic 1min, 0.22 μm of micropore filter
Membrane filtration, is obtained folate-targeted blank liposome, is designated as F-P-LP.F-P-LP is mixed with PEDF genes by upper table mass ratio, room
Temperature is incubated 30min, that is, the PEDF gene composites of modified with folic acid are obtained, and is designated as F-P-LP/PEDF.
Test example 1 expresses structure, amplification and the extraction purification of the plasmid of PEDF
(1) the structure operation of pAAV2-PEDF recombinant plasmids is as follows：By round pcr from plasmid pBLAST49-hPEDF
The total length PEDF gene cDNA fragments of human cloning, primer, upstream are designed according to PEDF gene orders：5'GGA ATT CAT GCA
GGC CCT GGT GCT ACT C 3'；Downstream：5'ACG CGT CGA CTT AGG GGC CCC TGG GGT CCA G 3',
This design of primers has I two restriction enzyme sites of EcoR I and Sal, respectively at 5' ends and 3' ends.Expressed with EcoR I and the digestions of Sal I and carried
Body pAAV2, the PEDF gene cDNA fragments after amplification through EcoR I and the digestions of Sal I after purification are used with the pAAV2 of linearisation
T4DNA ligases are connected, and obtain final product pAAV2-PEDF recombinant plasmids.Recombinant plasmid is transferred to Escherichia coli again, after amplification cultivation, is used
The a large amount of extracts kit extraction purification pAAV2-PEDF recombinant plasmids of high-purity plasmid.It is dense its to be determined with ultraviolet specrophotometer
Degree, the purity of pAAV2-PEDF, the pAAV2-PEDF of preparation are analyzed by the measure and agarose gel electrophoresis of A260/A280
260/A280=1.8~2.0；Detected through agarose gel electrophoresis simultaneously, dye-free body DNA, protein and RNA pollution, band
It is neat clear.
(2) with reference to the above method, from pVITRO2, pGenesil2.1, pGenesil2.4, pCMV-Script or
PBlast49 designs primer as carrier according to PEDF gene orders, from suitable enzyme digestion expression vector pVITRO2,
PGenesil2.1, pGenesil2.4, pCMV-Script or pBlast49, by after amplification through suitable enzyme digestion after purification
PEDF gene cDNA fragments with linearisation pVITRO2, pGenesil2.1, pGenesil2.4, pCMV-Script or
PBlast49 with DNA ligase connect, obtain final product pVITRO2-PEDF, pGenesil2.1-PEDF, pGenesil2.4-PEDF,
PCMV-Script-PEDF or pBlast49-PEDF recombinant plasmids.
Contrast pAAV2-PEDF, pVITRO2-PEDF, pGenesil2.1-PEDF, pGenesil2.4-PEDF, pCMV-
Script-PEDF or pBlast49-PEDF recombinant plasmids, nano-complex prepared by different recombinant plasmids is in particle diameter, zeta electricity
Position, envelop rate and release behaviour in vitro aspect are all without significant difference.Inventor is with pAAV2-PEDF, pVITRO2- simultaneously
PEDF, pGenesil2.1-PEDF, pGenesil2.4-PEDF, pCMV-Script-PEDF or pBlast49-PEDF recombinate matter
Grain, is prepared for compound respectively, and trial test research shows, transfection efficiency and internal tumor killing effect aspect do not have compound in vitro
There is significant difference, therefore have selected one of which recombinant plasmid i.e. pAAV2-PEDF has carried out transfection efficiency in vitro, internal tumor suppression
The researchs such as effect aspect.Hereinafter it is that the nano-complex prepared with pAAV2-PEDF recombinant plasmids carries out galenic pharmacy research.Examination
The plasmid and the mass ratio of modified with folic acid liposome for testing the expression of example 2 PEDF are screened
The plasmid and modified with folic acid liposome of expression PEDF are taken, by different quality ratio (1:0.5、1:1、1:2、1:4、1:6、
1:8、1:10、1:12、1:15、1:20) isometric mixing, obtains final product the dual-gene compositions of PEDF of modified with folic acid.By growth conditions
Good human breast cancer cell line Bcap-37 spreads six orifice plates, makes six orifice plate inner cell density about 40%-50% after inoculation, with containing 10%
The DMEM medium culture cells of FBS；When cell density is 60% or so, rinsed with the DMEM culture mediums without folic acid serum-free
Cell 2 times, adds 800 μ l without folic acid plasma-free DMEM medium, and above-mentioned composition is separately added into again (per hole after 37 DEG C of culture 1h
Plus about 2 μ g plasmids), after 37 DEG C are continued to cultivate 6h, protein expression situation is detected with Western blotting.Concrete operations position：
Cell culture medium is discarded, the fresh DMEM containing 10%FBS is added, 2ml/ holes after 37 DEG C are continued to cultivate 48h, discard culture medium；
100 μ l lysates are added per hole, then cell suspension is transferred in the aseptic Ep pipes of prior precooling；With cell crushing instrument ultrasound
5s, is spaced 5s, is repeated 2 times；Cell pyrolysis liquid after ultrasound is placed in into water-bath 5min in 100 DEG C of water-baths makes albuminous degeneration, so
Stand 5min on ice afterwards；2 μ l protein samples are taken in the concentration that albumen is determined on nucleic acid-protein quantitative instrument；To in gained protein sample
Albumen loading buffer are added, then is separated with SDS-PAGE proteins gel electrophoresis, transferring film, closing, immuning hybridization simultaneously develop the color.
Result shows：The mass ratio of the plasmid and modified with folic acid liposome of expressing PEDF is 1:1~1:When 20, obtained composition has
Protein expressioning product, but 1:0.5 does not almost detect protein expressioning product, 1:Expression product is relatively fewer when 2；1:20 group
Compound is larger to the toxicity of cell, and cell death is more, and 1:15 composition has mild toxicity, and cell has a little death.Therefore,
Consider gene expression efficiency and the aspect of cytotoxicity two, the plasmid of expression PEDF is with the mass ratio of modified with folic acid liposome most
It is defined as 1 eventually:1~1:15, preferably 1:2~1:15.
The consumption screening of the modified with folic acid matrix material of test example 3
Take different mol ratio example matrix material (folic acid accounts for TL molal quantity and is respectively：0th, 0.01%, 0.05%,
0.1%th, 0.25%, 0.5%, 1%, 2.5%, 5%, 8%) mix, modified with folic acid liposome is prepared using membrane process.Folic acid is accounted for
TL molal quantity is 0~5%, and liposome can be successfully obtained, and outward appearance is in light blue translucent colloidal solution, but up to 8%
When, obtained liposome colloidal solution is white without opalescence, and is precipitated more；Modified with folic acid lipid consumption is fat obtained in 5%
Plastid, after 4 DEG C are placed 2 weeks, has precipitation to generate, and stability is slightly poor, liposome stability prepared by other usage ratios compared with
It is good.Take the logarithm the MCF-7 Human Breast Cancer Cells bed board in growth period, after nutrient solution after adherent 24h, is discarded, add serum-free, without leaf
(folic acid accounts for TL molal quantity and is respectively the different liposome that sour DMEM dilutions are prepared：0th, 0.01%, 0.05%, 0.1%,
0.25%th, 0.5%, 1%, 4h 2.5%) is cultivated.Then the method for test example 2 is pressed, albumen table is detected with Western blotting
Up to situation.Result shows, not plus the liposome of modified with folic acid lipid has not detected protein expression, does not add 0.01% modified with folic acid fat
The liposome of matter has target protein to detect, but detected level is relatively fewer, adds the lipid of 0.05%~2.5% modified with folic acid lipid
Body group, target protein band is more visible.Therefore, shaping, stability and the gene expression efficiency of liposome, folic acid are considered
The modified with folic acid lipid consumption of modified liposome is ultimately determined to：Folic acid accounts for the 0.01~5% of TL molal quantity, preferably
0.05~2.5%.
Therapeutic actions of the test example 4F-P-LP/PEDF to human cervical carcinoma cell Hela Metastasis tumor of abdomen is studied
4~6 week old Balb/C Female nude mices intraperitoneal inoculations about 5 × 106Individual Hela cells/only, be randomly divided into after inoculation by
Mouse is randomly divided into 5 groups, i.e. 5% glucose (control), self-control FLP/PEDF, PLP/PEDF, FLP/PAAV2, PLP/
PAAV2 groups, every group 5.Every 2 days intraperitoneal injections once, it is administered 10 times altogether, dosage is PEDF or PAAV2250
μg/kg.Administration record each body weights of mouse of each group, are averaged every time every time, and Mouse Weight is painted according to statistics
Change curve.Nude mice is put to death, ascites is extracted out, ascites volume is recorded；The tumor tissues of nude mice are taken out, is counted in every nude mice abdominal cavity
Tumor nodule number simultaneously measures tumor nodule weight；Calculate tumor control rate.Result is shown in Fig. 2, Fig. 3 and Fig. 4.With control group and other
Test group is compared, and the PEDF compounds (FLP/PEDF) of modified with folic acid can substantially reduce tumor weight, enhancing tumour growth suppression
Rate (Fig. 2) processed, reduces ascites (Fig. 3), reduces belly cavity tumor node number (Fig. 4).Show, the PEDF compounds tool of modified with folic acid
There is preferable antitumor activity.
<110>Sichuan University
<120>The PEDF gene composites of tumour cell folacin receptor targeting
<140>
<141>
<160>1
<210>1
<211>418
<212>AA
<213>Artificial sequence
<220>
<221>misc_fiture
<440>1
1 mqalvlllci gallghsscq npasppeegs pdpdstgalv eeedpffkvp
vnklaaavsn
61 fgydlyrvrs smspttnvll splsvatals alslgadert esiihralyy
dlisspdihg
121 tykelldtvt apqknlksas rivfekklri kssfvaplek sygtrprvlt
gnprldlqei
181 nnwvqaqmkg klarstkeip deisilllgv ahfkgqwvtk fdsrktsled
fyldeertvr
241 vpmmsdpkav lrygldsdls ckiaqlpltg smsiifflpl kvtqnltlie
esltsefihd
301 idrelktvqa vltvpklkls yegevtkslq emklqslfds pdfskitgkp
ikltqvehra
361 gfewnedgag ttpspglqpa hltfpldyhl nqpfifvlrd tdtgallfig kildprgp
Claims (8)
1. PEDF gene composites of a kind of modified with folic acid, it is characterised in that：Plasmid and modified with folic acid fat containing coding PEDF
Plastid.
2. composition according to claim 1, it is characterised in that the plasmid and folic acid of described coding pedf protein gene
The mass ratio of modified liposome is 1:1~1:20.
3. pharmaceutical composition according to claim 1, it is characterised in that the plasmid of described coding pedf protein gene with
The mass ratio of modified with folic acid liposome is 1:2~1:15.
4. pharmaceutical composition according to claims 1 to 3, it is characterised in that the modified with folic acid liposome, contains lipid
Material and modified with folic acid lipid.
5. modified with folic acid lipid according to claim 4, folic acid accounts for the 0.01~5% of TL molal quantity, preferably
0.05~2.5%.
6. modified with folic acid lipid according to claim 4, is coupled with matrix material by folic acid and is formed.
7. in matrix material according to claim 4, including phosphatide and its derivative or cholesterol and its derivative one
Plant or various.
8. the pharmaceutical composition described in any one of claim 1 ~ 3 prepare treatment tumour medicine in purposes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610668056.3A CN106822920A (en) | 2016-08-15 | 2016-08-15 | The PEDF gene composites of tumour cell folacin receptor targeting |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610668056.3A CN106822920A (en) | 2016-08-15 | 2016-08-15 | The PEDF gene composites of tumour cell folacin receptor targeting |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106822920A true CN106822920A (en) | 2017-06-13 |
Family
ID=59145536
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610668056.3A Pending CN106822920A (en) | 2016-08-15 | 2016-08-15 | The PEDF gene composites of tumour cell folacin receptor targeting |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106822920A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109876156A (en) * | 2019-03-21 | 2019-06-14 | 四川大学 | Liposome complex of modified with folic acid and its preparation method and application |
| CN110693838A (en) * | 2019-11-26 | 2020-01-17 | 深圳职业技术学院 | Preparation of folic acid modified tetrapeptide YGLF-loaded core-shell type nanoliposome |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101804209A (en) * | 2010-03-31 | 2010-08-18 | 四川大学 | PEDF (Pigment Epithelial Derived Factor) gene PLGA nano compound as well as preparation method and use thereof |
| CN102579342A (en) * | 2012-02-16 | 2012-07-18 | 中国药科大学 | Targeting ginkgolide B solid lipid nanoparticle and preparation method thereof |
| CN105641714A (en) * | 2015-12-28 | 2016-06-08 | 四川大学 | Folic acid modified VEGFR2/Tie2 double gene composition |
-
2016
- 2016-08-15 CN CN201610668056.3A patent/CN106822920A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101804209A (en) * | 2010-03-31 | 2010-08-18 | 四川大学 | PEDF (Pigment Epithelial Derived Factor) gene PLGA nano compound as well as preparation method and use thereof |
| CN102579342A (en) * | 2012-02-16 | 2012-07-18 | 中国药科大学 | Targeting ginkgolide B solid lipid nanoparticle and preparation method thereof |
| CN105641714A (en) * | 2015-12-28 | 2016-06-08 | 四川大学 | Folic acid modified VEGFR2/Tie2 double gene composition |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109876156A (en) * | 2019-03-21 | 2019-06-14 | 四川大学 | Liposome complex of modified with folic acid and its preparation method and application |
| CN110693838A (en) * | 2019-11-26 | 2020-01-17 | 深圳职业技术学院 | Preparation of folic acid modified tetrapeptide YGLF-loaded core-shell type nanoliposome |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104922676B (en) | Polyamine derivatives | |
| JP6027592B2 (en) | Nucleic acid-lipopolymer composition | |
| CN104434806B (en) | Lipid-mixed poly (lactic-co-glycolic acid) (PLGA) nanoparticle having high drug loading amount and active targeting effect | |
| AU2009223313A1 (en) | Biodegradable cross-linked branched poly (alkylene imines) | |
| CN109837306A (en) | Contain the excretion body and its preparation method and application of miRNA-204-5p | |
| KR100794449B1 (en) | Composition of cationic phospho lipid nanoparticles for effective delivery of nucleic acids | |
| CN110522918A (en) | It targets element and preparation method thereof and uses | |
| CN105343895A (en) | Dual-targeting ursolic acid (UA)/siRNA loaded fluorescent mesoporous silica dioxide-hyaluronic acid and application | |
| CN102260356B (en) | Chitosan derivative used as gene vector, and preparation method and application thereof | |
| CN111632153B (en) | Chemical gene drug co-loaded targeting nano drug delivery system and preparation method thereof | |
| JP2022531010A (en) | Drug delivery carrier and pharmaceutical product using it | |
| CN103893124A (en) | Gene-therapy drug delivery system based on cooperative assembling | |
| CN102397554A (en) | Tumor-targeting double-drug carrying and delivery system and preparation method thereof | |
| CN111450061A (en) | Hybrid mesenchymal stem cell exosome drug delivery system and preparation method and application thereof | |
| CN106822920A (en) | The PEDF gene composites of tumour cell folacin receptor targeting | |
| CN105641714A (en) | Folic acid modified VEGFR2/Tie2 double gene composition | |
| CN103834035B (en) | A kind of cationization laminarin and its preparation method and application | |
| CN114053225B (en) | High-efficiency low-toxicity cationic liposome with good stability for gene delivery and application thereof | |
| CN101804209A (en) | PEDF (Pigment Epithelial Derived Factor) gene PLGA nano compound as well as preparation method and use thereof | |
| CN106188223B (en) | Compound containing dipeptide lipid cations and preparation method and application thereof | |
| CN110279673B (en) | AuNP @ PP/poly (I: C), preparation method thereof and application thereof in preparation of drugs for treating glioma | |
| CN113501889A (en) | Preparation method and application of pseudo-ginseng polysaccharide cationic derivative | |
| CN113058028A (en) | Preparation and application of composite nano-medicine | |
| CN102030754B (en) | Non-viral vector, and preparation method and application thereof | |
| CN108431224A (en) | A kind of small RNA and medical composition and its use |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170613 |
|
| RJ01 | Rejection of invention patent application after publication |