CN108030931A - A kind of application of medicinal liposome/p53 gene composites - Google Patents
A kind of application of medicinal liposome/p53 gene composites Download PDFInfo
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- CN108030931A CN108030931A CN201711363420.6A CN201711363420A CN108030931A CN 108030931 A CN108030931 A CN 108030931A CN 201711363420 A CN201711363420 A CN 201711363420A CN 108030931 A CN108030931 A CN 108030931A
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- resveratrol
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- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—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 'active' part of the composition delivered, i.e. the nucleic acid delivered
- A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
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- A61K9/00—Medicinal preparations characterised by special physical form
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Abstract
This divisional application provides a kind of application of medicinal liposome/p53 gene composites.The compound is made of the medicinal liposome containing resveratrol and p53 genes.First using polypeptide type cation lipoid, lipoid and resveratrol are helped as primary raw material, pass sequentially through film dispersion method, ultrasonic aquation method and gel chromatographic columns partition method, prepare medicinal liposome, then the medicinal liposome and p53 genes are mixed, medicinal liposome/gene composite is prepared by the compound method of electrostatic.Medicinal liposome/gene composite provided by the invention activates p38MAPK kinase activities by resveratrol and induces p53 gene activations, it is allowed to express wild-type p 53 protein, collaboration plays the antitumor action of resveratrol and p53 genes, triggers apoptosis of tumor cells, achievees the purpose that oncotherapy.Extracorporeal biology research shows that the compound has certain cell inhibitory effect ability, and wild-type p 53 protein expression is high, provides new approaches for oncotherapy, has potential application value.
Description
The application is application number 2017101315500,7 days March 2017 applying date, a kind of denomination of invention " medicine lipid
The divisional application of body/p53 gene composites and its preparation method and application ".
Technical field：
The present invention relates to a kind of medicinal liposome/p53 gene composites, more specifically to a kind of containing resveratrol
Polypeptide type cationic drug liposome preparation and its with p53 genes formed new antitumoral complex formulation in application,
Belong to therapeutic field of tumor new medicinal preparation and preparation method and application.
Background technology：
The transhipment treatment of the joint of medicine and gene is that cancer therapy drug and gene efficient are delivered to tumour by one kind using carrier
Among cell, medicine is discharged in due course and gene has reached the biomedical treatment method of synergistic treatment target.At present, joint transhipment
Therapy system mainly includes the treatment method system that medicine/medicine, medicine/siRNA, medicine/pDNA etc. are directed to various diseases.Fortune
The carrier lipid body of medicine and nucleic acid is carried, further includes the polymer carrier such as polymer micelle and PLGA nano-particles.In same load
In system system, joint transhipment medicine and DNA, which act on tumor cell ratio and individually transport DNA or medicine, more therapeutic effect.Cause
This, prepares the emphasis that a kind of safe and efficient complexes carrier has become Recent study.
China has abundant resources of medicinal plant, and especially ethnic mimority area natural plants species is various, at present, camptothecine,
A variety of natural anti-cancer drugs such as taxol, Rabdosia rubescens class, Cephalotaxines largely originate from this area.Natural antitumor medicine
Research and development and application are had been widely used in the treatment of cancer with the advantage of its hypotoxicity, less toxic side effect.Resveratrol conduct
The progress of a kind of newfound natural anti-cancer drugs for originating from China ethnic mimority area, its pharmacological activity and anticancer mechanism is just firm
Ground zero.At present, it has been found that resveratrol is a variety of to breast cancer, colon cancer, prostate cancer, oophoroma, cutaneum carcinoma, liver cancer etc.
Malignant cell has obvious inhibitory action.Wherein, growth of the resveratrol to hormone-dependent tumor cells has aobvious
The inhibitory action of work.From the structure of resveratrol, it is a kind of polyphenols of non-flavonoids, since oxidative damage is
Cause the one of the major reasons of cancer, the anti-oxidation function of polyphenol can play cancer prevention effect and suppression to a certain extent
Make and use.The anticancer mechanism of resveratrol is sufficiently complex, has now been found that, resveratrol can be lured by activating p38MAPK activity
The phosphorylation of the 15th serine of p53 albumen is so as to activate the activation of p53 albumen, the p53 albumen of phosphorylation in guided cell path
The expression of the apoptotic proteins such as Bcl-2, Bax can be lowered, Bax albumen can open mitochondrial membrane passage, and cytochrome c withers with other
Dying precursor protein can just discharge from mitochondria, and caspase activation, originates apoptosis.However, resveratrol light, heat and
Decomposition is oxidized easily under oxidant existence condition, limits its application in food and medicine industry to a certain extent.
With the development of molecular biology, people are known from the molecular level that tumour is caused a disease, and cancer is actually one
Kind of genopathy, otherwise it is the inactivation of tumor suppressor gene, otherwise it is proto-oncogene by abnormal activation.At present, main tumor suppressor gene bag
Include Rb genes, p53 genes, WT1 genes, DCC genes, apc gene, NF1 genes, MTS1 genes, WAF1 genes, BRCA genes and
BRCA2 genes etc..Wherein, p53 genes play a significant role on cell cycle and control Apoptosis is adjusted, and have research table
Bright, P53 genes play the role of controlling Apoptosis by expressing the p53 albumen of wild type, are ground in terms of having become tumour
The hot spot studied carefully.
Cationic-liposome can be effectively protected wrapped medicine as non-virus carrier material, improve its stability
And bioavilability, and also have that toxicity is low, biocompatibility is high and a clear superiority such as biodegradable.In medicine and gene
In terms of joint transhipment, Sadd etc. carries the siRNA and adriamycin of silence Bcl-2, MDP1, MDR1 gene by cationic-liposome altogether
(DOX) lung carcinoma cell is treated, and demonstrates the synergistic therapeutic action of gene and medicine；Kang etc. similarly by sun from
Sub- liposome carries NIK-siRNA and 5 FU 5 fluorouracil (5-FU) treatment liver cancer altogether, the results show that the tumour cell that liposome carries altogether
Apoptosis situation is significantly more than single carry.Cationic-liposome can mediate gene to be conveyed into cell as pharmaceutical carrier, have
Unique advantage and good development prospect.
The content of the invention
In order to solve deficiency of the prior art, the present invention provides a kind of medicinal liposome/gene for oncotherapy
Compound, the compound are made of the medicinal liposome containing resveratrol and p53 genes.The present invention also provides the medicine lipid
The preparation method of body/gene composite.First using polypeptide type cation lipoid, lipoid and resveratrol are helped as primary raw material, according to
It is secondary by film dispersion method, ultrasonic aquation method and gel chromatographic columns partition method, medicinal liposome is prepared, then by the medicine lipid
Body is mixed with p53 genes, and medicinal liposome/gene composite is prepared by the compound method of electrostatic.
The purpose of the present invention is what is be achieved through the following technical solutions：A kind of medicinal liposome/p53 gene composites, should
Compound is made of medicinal liposome and p53 genes, and the mass ratio of medicinal liposome and p53 genes is 1:1~16:1.
Preferably, the mass ratio of the medicinal liposome and p53 genes is 2:1~8:1.
The preparation method of the medicinal liposome, concrete operation step are as follows：
Polypeptide type cation lipoid and resveratrol are dissolved in organic solvent, addition helps lipoid fully to dissolve, polypeptide
Type cation lipoid, resveratrol are 5 with helping the mass ratio of lipoid:1:1~200:1:1, whirlpool shakes 1-20 seconds and mixes after dissolving
Close uniform, with inert gas blown film 10-60min, 1-48h be dried in vacuo in 25-120 DEG C, add water in 25-45 DEG C of ultrasound,
The aqueous solution that concentration is 0.5-5mg/mL is configured to, through sephadex post separation after centrifugation, obtains medicinal liposome.
The organic solvent is the one or more in methanol, chloroform and ethyl acetate.
Another object of the present invention is that the preparation method of medicinal liposome/p53 gene composites, concrete operations are claimed
Step is as follows：
Medicinal liposome is mixed with p53 genes, vortex, which shakes 1-20 seconds, to be uniformly mixed, in 20 DEG C of -40 DEG C of incubation 5-
60min, is obtained by the compound preparation method of electrostatic.
Described helps lipoid as the one or more in DOPE, cholesterol, lecithin.
Preferably, the lipoid that helps is DOPE.
Preferably, the polypeptide type cation lipoid is CDO12, CDO14, CDO16.
3rd purpose of the invention is medicinal liposome/p53 gene composites to be claimed and said medicine liposome exists
Prepare the application in anti-tumor medicine.
Medicinal liposome provided by the invention/p53 gene composites activate p38MAPK kinase activities by resveratrol and lure
P53 gene activations are led, are allowed to express wild-type p 53 protein, collaboration plays the antitumor action of resveratrol and p53 genes, draws
Swell apoptosis of tumor, achieve the purpose that oncotherapy.Extracorporeal biology research shows that the compound cytotoxicity is low, wild
Type p53 protein expressions are high, provide new approaches for oncotherapy, have great clinical value.
Brief description of the drawings
Fig. 1 is the transmission electron microscope picture of resveratrol liposome, wherein (A) is CDO14-RSV20:1, (B) is CDO14-RSV
+D 10:1, (C) is CDO14-RSV100:1；
Fig. 2 is resveratrol liposome encapsulation testing result；
Fig. 3 is the electrophoresis retardation lab diagram of resveratrol liposome/P53 gene composites, wherein the 1st swimming lane is
Marker (λ DNA/EcoR I+Hind III Markers), the 2nd swimming lane is naked p53 genes, and the 3rd~9 swimming lane is respectively white lamb's-quarters
Reed alcohol liposome and/p53 gene composite qualities are than 1:1、2:1、3:1、4:1、6:1、8:1；
Fig. 4 A are resveratrol liposome/p53 gene composites expression effect figure in Hela cells；
Fig. 4 B are resveratrol liposome/p53 gene composites expression effect figure in MCF-7 cells；
Fig. 5 A are resveratrol liposome/p53 gene composite induced Hcla cell apoptosis figures；
Fig. 5 B induce MCF-7 Apoptosis figures for resveratrol liposome/p53 gene composites；
Fig. 6 A are resveratrol liposome/influence of the P53 gene composites to Hela cell survival rates；
Fig. 6 B are resveratrol liposome/influence of the P53 gene composites to MCF-7 cell survival rates.
Embodiment：
The present invention is described in detail below by embodiment, but is not used in and limits the scope of the invention, such as without spy
Different explanation, chemical reagent and medicine involved in the present invention are commercially available, and unless otherwise specified, involved method is routine
Method.
Embodiment 1
Cation lipoid CDO14 and resveratrol are dissolved in the organic solvent of methanol and chloroform, addition helps lipoid
DOPE fully dissolves, and cation lipoid, resveratrol are 5 with helping the mass ratio of lipoid:1:1, whirlpool concussion afterwards mixes for 10 seconds
Uniformly, inert gas blown film 10min, 25 DEG C of vacuum drying 12h, adds water in 25 DEG C of ultrasounds, it is 0.5mg/ to be formulated as concentration
The aqueous solution of mL, through sephadex post separation after centrifugation, obtains medicinal liposome, then by medicinal liposome and p53 genes
Mixing, the mass ratio of medicinal liposome and p53 genes is 1:1, vortex, which shakes 10 seconds, to be uniformly mixed, and 20 DEG C of incubation 10min, pass through
The compound preparation method of electrostatic obtains.
Embodiment 2
Cation lipoid CDO14 and resveratrol are dissolved in the organic solvent of methanol, addition helps lipoid cholesterol to fill
Divide dissolving, cation lipoid, resveratrol are 10 with helping the mass ratio of lipoid:1:1, whirlpool concussion afterwards is uniformly mixed for 15 seconds,
Inert gas blown film 15min, 30 DEG C of vacuum drying 18h, add water in 30 DEG C of ultrasounds, are formulated as the water that concentration is 1.0mg/mL
Solution, through sephadex post separation after centrifugation, obtains medicinal liposome, then mixes medicinal liposome with p53 genes, medicine
The mass ratio of composite lipidosome and p53 genes is 2:1, vortex, which shakes 15 seconds, to be uniformly mixed, and 25 DEG C of incubation 15min, are answered by electrostatic
The preparation method of conjunction obtains.
Embodiment 3
Cation lipoid CDO12 and resveratrol are dissolved in the organic solvent of ethyl acetate, addition helps lipoid DOPE
Fully dissolving, cation lipoid, resveratrol are 20 with helping the mass ratio of lipoid:1:1, the mixing in 20 seconds of whirlpool concussion afterwards is equal
Even, inert gas blown film 20min, 40 DEG C of vacuum drying 24h, add water in 35 DEG C of ultrasounds, it is 1.5mg/mL to be formulated as concentration
Aqueous solution, through sephadex post separation after centrifugation, obtain medicinal liposome, then mix medicinal liposome and p53 genes
Close, the mass ratio of medicinal liposome and p53 genes is 3:1, vortex, which shakes 20 seconds, to be uniformly mixed, 30 DEG C of incubation 20min, by quiet
The preparation method for replying conjunction by cable obtains.
Embodiment 4
Cation lipoid CDO16 and resveratrol are dissolved in the organic solvent of ethyl acetate, addition helps lipoid lecithin
Fat fully dissolves, and cation lipoid, resveratrol are 100 with helping the mass ratio of lipoid:1:1, the mixing in 5 seconds of whirlpool concussion afterwards is equal
Even, inert gas blown film 25min, 45 DEG C of vacuum drying 30h, add water in 40 DEG C of ultrasounds, it is 2.0mg/mL to be formulated as concentration
Aqueous solution, through sephadex post separation after centrifugation, obtain medicinal liposome, then mix medicinal liposome and p53 genes
Close, the mass ratio of medicinal liposome and p53 genes is 4:1, vortex, which shakes 5 seconds, to be uniformly mixed, 35 DEG C of incubation 25min, by quiet
The preparation method for replying conjunction by cable obtains.
Embodiment 5
Cation lipoid CDO16 and resveratrol are dissolved in the organic solvent of methanol, addition helps lipoid cholesterol to fill
Divide dissolving, cation lipoid, resveratrol are 200 with helping the mass ratio of lipoid:1:1, whirlpool concussion afterwards is uniformly mixed for 12 seconds,
Inert gas blown film 30min, 50 DEG C of vacuum drying 36h, add water in 45 DEG C of ultrasounds, are formulated as the water that concentration is 2.5mg/mL
Solution, through sephadex post separation after centrifugation, obtains medicinal liposome, then mixes medicinal liposome with p53 genes, medicine
The mass ratio of composite lipidosome and p53 genes is 6:1, vortex, which shakes 10 seconds, to be uniformly mixed, and 40 DEG C of incubation 30min, are answered by electrostatic
The preparation method of conjunction obtains.
Embodiment 6
Cation lipoid CDO14 and resveratrol are dissolved in the organic solvent of chloroform, addition helps lipoid lecithin to fill
Divide dissolving, cation lipoid, resveratrol are 5 with helping the mass ratio of lipoid:1:1, whirlpool concussion afterwards is uniformly mixed for 15 seconds, lazy
Property gas blown film 35min, 50 DEG C of vacuum drying 40h, add water in 30 DEG C of ultrasounds, it is the water-soluble of 3.0mg/mL to be formulated as concentration
Liquid, through sephadex post separation after centrifugation, obtains medicinal liposome, then mixes medicinal liposome with p53 genes, medicine
The mass ratio of liposome and p53 genes is 8:1, vortex, which shakes 20 seconds, to be uniformly mixed, and 25 DEG C are incubated 20min, compound by electrostatic
Preparation method obtain.
Embodiment 7
Cation lipoid CDO14 and resveratrol are dissolved in the organic solvent of chloroform, methanol and ethyl acetate, added
Lipoid lecithin is helped fully to dissolve, cation lipoid, resveratrol are 10 with helping the mass ratio of lipoid:1:1, whirlpool concussion afterwards
It is uniformly mixed within 10 seconds, inert gas blown film 40min, 60 DEG C of vacuum drying 48h, add water in 40 DEG C of ultrasounds, be formulated as concentration
For the aqueous solution of 3.5mg/mL, through sephadex post separation after centrifugation, obtain medicinal liposome, then by medicinal liposome with
P53 genes mix, and the mass ratio of medicinal liposome and p53 genes is 2:1, vortex, which shakes 15 seconds, to be uniformly mixed, 40 DEG C of incubations
15min, is obtained by the compound preparation method of electrostatic.
Embodiment 8
Cation lipoid CDO12 and resveratrol are dissolved in the organic solvent of ethyl acetate, addition helps lipoid DOPE
Fully dissolving, cation lipoid, resveratrol are 100 with helping the mass ratio of lipoid:1:1, the mixing in 15 seconds of whirlpool concussion afterwards is equal
Even, inert gas blown film 20min, 80 DEG C of vacuum drying 24h, add water in 45 DEG C of ultrasounds, it is 4.0mg/mL to be formulated as concentration
Aqueous solution, through sephadex post separation after centrifugation, obtain medicinal liposome, then mix medicinal liposome and p53 genes
Close, the mass ratio of medicinal liposome and p53 genes is 4:1, vortex, which shakes 20 seconds, to be uniformly mixed, 25 DEG C of incubation 30min, by quiet
The preparation method for replying conjunction by cable obtains.
Embodiment 9
Cation lipoid CDO16 and resveratrol are dissolved in the organic solvent of methanol, addition helps lipoid cholesterol to fill
Divide dissolving, cation lipoid, resveratrol are 20 with helping the mass ratio of lipoid:1:1, whirlpool concussion afterwards is uniformly mixed for 20 seconds,
Inert gas blown film 50min, 90 DEG C of vacuum drying 12h, add water in 25 DEG C of ultrasounds, are formulated as the water that concentration is 4.5mg/mL
Solution, through sephadex post separation after centrifugation, obtains medicinal liposome, then mixes medicinal liposome with p53 genes, medicine
The mass ratio of composite lipidosome and p53 genes is 6:1, vortex, which shakes 10 seconds, to be uniformly mixed, and 30 DEG C of incubation 60min, are answered by electrostatic
The preparation method of conjunction obtains.
Embodiment 10
Cation lipoid CDO14 and resveratrol are dissolved in the organic solvent of methanol and chloroform, addition helps lipoid
DOPE fully dissolves, and cation lipoid, resveratrol are 5 with helping the mass ratio of lipoid:1:1, the mixing in 3 seconds of whirlpool concussion afterwards is equal
Even, inert gas blown film 60min, 100 DEG C of vacuum drying 8h, add water in 30 DEG C of ultrasounds, it is 5.0mg/mL to be formulated as concentration
Aqueous solution, through sephadex post separation after centrifugation, obtain medicinal liposome, then mix medicinal liposome and p53 genes
Close, the mass ratio of medicinal liposome and p53 genes is 8:1, vortex, which shakes 20 seconds, to be uniformly mixed, 40 DEG C of incubation 20min, by quiet
The preparation method for replying conjunction by cable obtains.
Embodiment 11
Cation lipoid CDO14 and resveratrol are dissolved in the organic solvent of methanol and ethyl acetate, addition helps class
Fat DOPE fully dissolves, and cation lipoid, resveratrol are 100 with helping the mass ratio of lipoid:1:1, whirlpool concussion afterwards 10 seconds
It is uniformly mixed, inert gas blown film 20min, 120 DEG C of vacuum drying 4h, add water in 25 DEG C of ultrasounds, being formulated as concentration is
The aqueous solution of 2.0mg/mL, through sephadex post separation after centrifugation, obtains medicinal liposome, then by medicinal liposome with
P53 genes mix, and the mass ratio of medicinal liposome and p53 genes is 6:1, vortex, which shakes 15 seconds, to be uniformly mixed, 25 DEG C of incubations
15min, is obtained by the compound preparation method of electrostatic.
Embodiment 12
Cation lipoid CDO16 and resveratrol are dissolved in the organic solvent of chloroform, addition helps lipoid cholesterol to fill
Divide dissolving, cation lipoid, resveratrol are 200 with helping the mass ratio of lipoid:1:1, whirlpool concussion afterwards is uniformly mixed for 15 seconds,
Inert gas blown film 10min, 40 DEG C of vacuum drying 20h, add water in 30 DEG C of ultrasounds, are formulated as the water that concentration is 1.0mg/mL
Solution, through sephadex post separation after centrifugation, obtains medicinal liposome, then mixes medicinal liposome with p53 genes, medicine
The mass ratio of composite lipidosome and p53 genes is 1:1, vortex, which shakes 20 seconds, to be uniformly mixed, and 40 DEG C of incubation 15min, are answered by electrostatic
The preparation method of conjunction obtains.
Embodiment 13
Cation lipoid CDO12 and resveratrol are dissolved in the organic solvent of chloroform, ethyl acetate and methanol, added
Lipoid lecithin is helped fully to dissolve, cation lipoid, resveratrol are 10 with helping the mass ratio of lipoid:1:1, whirlpool concussion afterwards
It is uniformly mixed within 20 seconds, inert gas blown film 15min, 70 DEG C of vacuum drying 18h, add water in 40 DEG C of ultrasounds, be formulated as concentration
For the aqueous solution of 3.0mg/mL, through sephadex post separation after centrifugation, obtain medicinal liposome, then by medicinal liposome with
P53 genes mix, and the mass ratio of medicinal liposome and p53 genes is 3:1, vortex, which shakes 15 seconds, to be uniformly mixed, 35 DEG C of incubations
30min, is obtained by the compound preparation method of electrostatic.
Embodiment 14
Cation lipoid CDO14 and resveratrol are dissolved in the organic solvent of chloroform, addition helps lipoid cholesterol to fill
Divide dissolving, cation lipoid, resveratrol are 20 with helping the mass ratio of lipoid:1:1, whirlpool concussion afterwards is uniformly mixed for 1 second, lazy
Property gas blown film 20min, 30 DEG C of vacuum drying 48h, add water in 25 DEG C of ultrasounds, it is the water-soluble of 0.5mg/mL to be formulated as concentration
Liquid, through sephadex post separation after centrifugation, obtains medicinal liposome, then mixes medicinal liposome with p53 genes, medicine
The mass ratio of liposome and p53 genes is 6:1, vortex, which shakes 20 seconds, to be uniformly mixed, and 40 DEG C are incubated 10min, compound by electrostatic
Preparation method obtain.
Embodiment 15
Cation lipoid CDO16 and resveratrol are dissolved in the organic solvent of methanol, addition helps lipoid DOPE abundant
Dissolving, cation lipoid, resveratrol are 100 with helping the mass ratio of lipoid:1:1, whirlpool concussion afterwards is uniformly mixed for 10 seconds, lazy
Property gas blown film 40min, 100 DEG C of vacuum drying 3h, add water in 40 DEG C of ultrasounds, it is the water-soluble of 1.5mg/mL to be formulated as concentration
Liquid, through sephadex post separation after centrifugation, obtains medicinal liposome, then mixes medicinal liposome with p53 genes, medicine
The mass ratio of liposome and p53 genes is 1:1, vortex, which shakes 20 seconds, to be uniformly mixed, and 20 DEG C are incubated 15min, compound by electrostatic
Preparation method obtain.
Embodiment 16
Cation lipoid CDO12 and resveratrol are dissolved in the organic solvent of ethyl acetate, addition helps lipoid courage to consolidate
Alcohol fully dissolves, and cation lipoid, resveratrol are 5 with helping the mass ratio of lipoid:1:1, the mixing in 20 seconds of whirlpool concussion afterwards is equal
Even, inert gas blown film 10min, 25 DEG C of vacuum drying 24h, add water in 45 DEG C of ultrasounds, it is 4.5mg/mL to be formulated as concentration
Aqueous solution, through sephadex post separation after centrifugation, obtain medicinal liposome, then mix medicinal liposome and p53 genes
Close, the mass ratio of medicinal liposome and p53 genes is 2:1, vortex, which shakes 10 seconds, to be uniformly mixed, 25 DEG C of incubation 20min, by quiet
The preparation method for replying conjunction by cable obtains.
Embodiment 17
Cation lipoid CDO14 and resveratrol are dissolved in the organic solvent of ethyl acetate and methanol, addition helps class
Fat lecithin fully dissolves, and cation lipoid, resveratrol are 10 with helping the mass ratio of lipoid:1:1, whirlpool concussion afterwards 15 seconds
It is uniformly mixed, inert gas blown film 20min, 30 DEG C of vacuum drying 48h, add water in 25 DEG C of ultrasounds, being formulated as concentration is
The aqueous solution of 2.0mg/mL, through sephadex post separation after centrifugation, obtains medicinal liposome, then by medicinal liposome with
P53 genes mix, and the mass ratio of medicinal liposome and p53 genes is 3:1, vortex, which shakes 20 seconds, to be uniformly mixed, 40 DEG C of incubations
5min, is obtained by the compound preparation method of electrostatic.
Embodiment 18
Cation lipoid CDO12 and resveratrol are dissolved in the organic solvent of chloroform and methanol, addition helps lipoid
DOPE fully dissolves, and cation lipoid, resveratrol are 100 with helping the mass ratio of lipoid:1:1, whirlpool concussion afterwards mixes for 20 seconds
Close uniformly, inert gas blown film 30min, 60 DEG C of vacuum drying 12h, add water in 30 DEG C of ultrasounds, being formulated as concentration is
The aqueous solution of 1.0mg/mL, through sephadex post separation after centrifugation, obtains medicinal liposome, then by medicinal liposome with
P53 genes mix, and the mass ratio of medicinal liposome and p53 genes is 8:1, vortex, which shakes 15 seconds, to be uniformly mixed, 40 DEG C of incubations
20min, is obtained by the compound preparation method of electrostatic.
Embodiment 19
Cation lipoid CDO16 and resveratrol are dissolved in the organic solvent of methanol and ethyl acetate, addition helps class
Fat DOPE fully dissolves, and cation lipoid, resveratrol are 5 with helping the mass ratio of lipoid:1:1, whirlpool concussion afterwards mixes for 20 seconds
Close uniformly, inert gas blown film 20min, 30 DEG C of vacuum drying 18h, add water in 30 DEG C of ultrasounds, being formulated as concentration is
The aqueous solution of 5.0mg/mL, through sephadex post separation after centrifugation, obtains medicinal liposome, then by medicinal liposome with
P53 genes mix, and the mass ratio of medicinal liposome and p53 genes is 2:1, vortex, which shakes 20 seconds, to be uniformly mixed, 30 DEG C of incubations
10min, is obtained by the compound preparation method of electrostatic.
Embodiment 20
Cation lipoid CDO12 and resveratrol are dissolved in the organic solvent of chloroform, addition helps lipoid cholesterol to fill
Divide dissolving, cation lipoid, resveratrol are 10 with helping the mass ratio of lipoid:1:1, whirlpool concussion afterwards is uniformly mixed for 10 seconds,
Inert gas blown film 40min, 55 DEG C of vacuum drying 20h, add water in 35 DEG C of ultrasounds, are formulated as the water that concentration is 1.0mg/mL
Solution, through sephadex post separation after centrifugation, obtains medicinal liposome, then mixes medicinal liposome with p53 genes, medicine
The mass ratio of composite lipidosome and p53 genes is 3:1, vortex, which shakes 20 seconds, to be uniformly mixed, and 20 DEG C of incubation 30min, are answered by electrostatic
The preparation method of conjunction obtains.
Embodiment 21
Present invention selection cervical cancer cell Hela cells and breast cancer cell MCF-7 cells are research object, will be encoded
The plasmid of green fluorescence protein gene and medicine lipid bluk recombination, utilize the table of inverted fluorescence microscope observation green fluorescent protein
Up to situation, it can directly reflect whether compound is successfully entered cell and the effect expressed.
As can be known from Fig. 4, the egfp expression level in Hela cells and MCF-7 cells is higher, illustrates this hair
Bright compound can successfully by cellular uptake and in cell, height be expressed, and has that further researches and develops to apply valency
Value.
Embodiment 22
Hoechst33342 be it is a kind of can be with the blue fluorescent dyes of penetration cell film, this hair relatively low to the toxicity of cell
Bright use cervical cancer cell Hela cells and breast cancer cell MCF-7 cells are main cell line, utilize Hoechst33342
Cell is dyed respectively, fluorescence intensity after Hoechst33342 is dyed is observed by inverted fluorescence microscope and observes cell
The state of form and nucleus, and then reflect the influence of complexes upon cell apoptosis.
As can be known from Fig. 5, cell is in bright blue fluorescence under inverted fluorescence microscope, and fluorescence intensity is stronger, and cell
Shade a little, only stretched in part, have fraction floats cell, attached cell form heterogeneity, cell density is relatively low, cell volume
It is rounded and diminishes and deform, cell edges obscures, and examine the state of nucleus, obvious apoptosis, occurs in nucleus shrinkage obfuscation
The outstanding feature feature in later stage, illustrates that medicinal liposome/gene composite of the present invention has and promotes Hela cells and MCF-7
The effect of Apoptosis.
Embodiment 23
Contain succinate dehydrogenase in the mitochondria of living cells, and do not have in dead cell, MTT can be by the amber in living cells
Amber acidohydrogenase reduction generation water-insoluble bluish violet first a ceremonial jade-ladle, used in libation, the present invention utilize this property, with MTT to Hela cells with
MCF-7 cells dye respectively, are detected with microplate reader, can reflect quantity of the living cells relative to dead cell indirectly, and then judge
Proliferation Ability ability of the compound to tumour cell.
As can be known from Fig. 6, resveratrol liposome with the increase with p53 gene compositely proportionals, cell survival rate it is gradual
Reduce, and when medicinal liposome and p53 genes mass ratio are 8/1, cell survival rate is minimum.Secondly, CDO14 blank is added
The cell survival rate of liposome is higher than medicinal liposome, and the cell survival rate for adding medicinal liposome is higher than compound.Then, it is empty
For white liposome in 1/1~8/1 compound ratio of liposome and p53 genes, cell survival rate is above 90%, and and commodity
Change reagent Lipofectamine 2000 to compare with DOTAP, blank liposome toxicity is suitable, even lower, illustrates the present invention's
Compound is respectively provided with certain cell inhibitory effect ability to MCF-7 cells and Hela cells.
Claims (1)
1. the application of a kind of medicinal liposome/p53 gene composites, it is characterised in that medicinal liposome and p53 genes are pressed into matter
Measure ratio 1:1~8:1 mixing, vortex, which shakes 1-20 seconds, to be uniformly mixed, and 5-60min is incubated in 20 DEG C -40 DEG C, compound by electrostatic
Preparation method obtains medicinal liposome/p53 gene composites, and prepared by medicinal liposome/p53 gene composites anti-cervix cancer
Cell Hela cells and breast cancer cell MCF-7 cell drugs.
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