CN105521497B - Embedded cationic liposomal gene carrier system of sucrose fatty ester and its preparation method and application - Google Patents
Embedded cationic liposomal gene carrier system of sucrose fatty ester and its preparation method and application Download PDFInfo
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- 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
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
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- 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
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
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- 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
- A61K48/0033—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 the non-active part being non-polymeric
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- 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
- A61K48/0041—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 the non-active part being polymeric
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- 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
A kind of embedded cationic liposomal gene carrier system of sucrose fatty ester and its preparation method and application, the gene vector system are the compounds being made of cation lipoid and the liposome for helping lipoid sucrose ester to prepare and genetic stew.Wherein cation lipoid includes peptide type cation lipoid, Gemini type cationic lipoid and quaternary ammonium salt cationic lipoid；Sucrose ester includes sucrose stearate, sucrose palmitate, Surfhope SE Cosme C 1216, sucrose oleate and sucrose erucate.The carrier can be effectively compressed Plasmid DNA and siRNA, in vitro in vivo can high-efficiency transfection, and toxicity is nearly free to cell and mouse.The embedded cationic liposomal gene carrier system of sucrose fatty ester of the present invention has research and application prospect well in gene therapy.
Description
Technical field
The invention belongs to field of biotechnology, especially sucrose fatty ester embedded cationic liposomal gene carrier systems
System and preparation method thereof.
Technical background
Gene therapy (gene therapy), which refers to, imports target cell for external source normal gene, to correct or compensate Yin Jiyin
Defect and abnormal caused disease, to reach therapeutic purposes.Nineteen ninety National Institutes of Health uses gene therapy method
Adenosine deaminase deficiency disease is treated, gene therapy is applied to clinic for the first time and achieves success.Use base within 2000
Because for the treatment of method successful treatment combined immunodeficiency syndrome, the validity of strategies in gene therapy is shown.Gene therapy will obtain
To further development, it is necessary to overcome the problems, such as three aspects: one, having the target gene for treatment and receive target gene
Cell；Two, the Modulatory character of gene expression is realized；Three, the efficient Gene delivery system with targeting is obtained.Wherein,
With the efficient Gene delivery system of targeting, i.e. genophore becomes the key factor for restricting gene therapy development.
Genophore mainly has two major classes: viral genetic vector, non-viral gene vector.Since being successfully made base for the first time
Because treatment clinical test since, most of viral vectors high using transfection efficiency, but viral vectors has very high exempt from
Epidemic focus is easy to recombinate with wild-type virus, and carrying capacity is limited, high production cost and easily contaminated.University of Pennsylvania of the U.S. in 1999
18 years old patients die is caused in clinical test by the gene therapy medicament of carrier of recombinant virus.In France in 2000
Ke Er hospital applies retroviral vector during treating severe combined immunodeficiency, has several in discovery in 2003
Children produce leukaemia.Therefore the development of viral vectors is restricted.Non-viral gene vector and viral genetic vector phase
Than having low immunogenicity, low toxicity, foreign DNA intergration rate low, no gene insert although transfection efficiency is relatively low
The advantages such as size limitation can even deliver nucleus or chromosome, using simple, and preparation facilitates.More and more researchers
Start the research in terms of selecting non-virus carrier to carry out gene therapy.Cationic-liposome is to study most thorough, technology at present most
Mature, most widely used non-viral carrier systems, the characteristic with class eucaryotic cell structure and biomembrane is degradable in vivo, can
To protect the bioactivity of its passenger gene segment.But there is also certain limitations for it, if cytotoxicity is big, to the target of organ
Tropism is unobvious, and gene delivery mechanism is indefinite etc., limits the development of cationic-liposome.Therefore, people have been devoted to
Various buildings and modification to cationic-liposome, it is intended to seek a kind of gene therapy medicament of high-efficiency low-toxicity.
Sucrose ester is a kind of nonionic surfactant, the simple substance generated by sucrose and fatty acid through esterification or mixing
Object.Sucrose ester has extensive hydrophilic lipophilic balance (HLB), has good dispersion, characteristic of solubilizing.Therefore, sucrose ester is done
The dispersibility of liposome can be enhanced to help lipoid to be used to prepare liposome, enhance the compatibility effect of liposome and drug, accelerate
Drug releasing rate promotes dispersion, prevents crystalline deposit, and extending the drug quality guarantee period.In addition, sucrose ester biocompatibility,
Biological degradability is strong, and the toxicity of liposome can be reduced by being used to prepare cationic-liposome.
Summary of the invention
That the object of the present invention is to provide a kind of cytotoxicities is small, the high sucrose fat of efficiency gene transfection in vitro and in vivo
Embedded cationic liposomal gene carrier system of acid esters and preparation method thereof and its application, overcome the deficiencies in the prior art.
The embedded cationic liposomal gene carrier system of sucrose fatty ester of the invention is embedded in by sucrose fatty ester
Formula cationic-liposome and genetic stew form, and the embedded cationic-liposome of the sucrose fatty ester is by cation lipoid
It is constituted with sucrose fatty ester.
The embedded cationic liposomal gene carrier system of sucrose fatty ester of the invention, wherein the sucrose-fatty
Ester is monoesters or dibasic acid esters, and wherein monoesters structure is as follows:
,
Wherein, R is selected from stearic acid or lauric acid or oleic acid or palmitinic acid or nutmeg acid or erucic acid；
The hydrophilic lipophilic balance of the sucrose fatty ester are as follows: HLB < 1 or 1≤HLB≤16.
The embedded cationic liposomal gene carrier system of sucrose fatty ester of the invention, wherein the cation lipoid
For peptide type cation lipoid, structural formula are as follows:
；
Or Gemini type cationic lipoid, structural formula are as follows:
；
Or quaternary ammonium salt cationic lipoid, structural formula are as follows:
；
Wherein: x is selected from 1~6；Y is selected from 1~8；R is selected from C8-20 Alkyl, the alkyl include straight chained alkyl and branched alkyl；
AA is selected from arginine or histidine or aspartic acid or alanine or glycine or ornithine or lysine；B is selected from Orn or Lys.
The embedded cationic liposomal gene carrier system of sucrose fatty ester of the invention, wherein the cationic
The mass ratio of rouge and sucrose fatty ester is 1 ︰, 8~8 ︰ 1；The embedded cationic-liposome of sucrose fatty ester and gene object
The mass ratio of matter is 0.5 ︰, 1 ~ 32 ︰ 1.
The embedded cationic liposomal gene carrier system of sucrose fatty ester of the invention, wherein the genetic stew
For Plasmid DNA or siRNA.
The preparation method of the embedded cationic liposomal gene carrier system of sucrose fatty ester of the invention, step is such as
Under:
The preparation of the embedded cationic-liposome of sucrose fatty ester:
(1) cation lipoid and sucrose fatty ester are dissolved in chloroform or methanol according to mass ratio 1:8~8:1 ratio
Obtain solution；
(2) solution for obtaining (1) step blows out uniform film under a nitrogen, is dried in vacuo 2~12h；
(3) use ethyl alcohol or water or physiological saline or phosphoric acid as buffer or in ethyl alcohol, water, physiological saline, phosphoric acid
The mixed liquor of the quality such as any two kinds carries out aquation, and hydration temperature is 10~80 DEG C；Hydration time is 1~10h, and ultrasonic vibration is extremely
Clarification, obtains the embedded cationic-liposome of sucrose fatty ester, and concentration is 0.5~3.0mg/mL；The sucrose fatty ester is embedding
Entering formula cationic-liposome is to be dispersed in the surface of the stable homogeneous that the particle size formed in water phase is 150~250nm to have
The cationic-liposome of positive charge；
The embedded sun of sucrose fatty ester is prepared using the embedded cationic-liposome of sucrose fatty ester and genetic stew
Cationic liposomal gene vector system:
By the embedded cationic-liposome of sucrose fatty ester and Plasmid DNA or siRNA, pass through electrostatic interaction
Form the nano particle for being dispersed in stable homogeneous in water phase, the specific steps are as follows:
(1) the embedded cationic-liposome of sucrose fatty ester is taken to be dispersed in cell culture fluid DMEM or RPMI1640,
It mixes, makes the 0.01 μ g/ μ μ g/ μ of L~0.64 L of concentration；
(2) 0.5~1.0 μ g Plasmid DNA or siRNA are diluted in cell culture fluid DMEM or RPMI1640,
It mixes, makes 0.02 μ g/ μ L of plasmid concentration；
(3) according to the embedded cationic-liposome of sucrose fatty ester and genetic stew mass ratio 0.5:1~32:1 ratio
(1) and (2) two dilutions are mixed, are placed at room temperature for 10 ~ 40min, the embedded cationic lipid of sucrose fatty ester can be obtained by example
Plastogene carrier system.
The sugarcane of the preparation method preparation of the embedded cationic liposomal gene carrier system of sucrose fatty ester of the invention
The embedded cationic liposomal gene carrier system of sugar fatty acid ester in cell transfecting application and in vivo transfection in answering
With specifically including that
1, the embedded cationic liposomal gene carrier of the sucrose ester can enter in cancer cell, complete target gene thin
Transfection intracellular.
2, the cell is human cervical carcinoma cell (Hela) and non-small lung adenocarcinoma cell (A549).It is combined under different N/P ratios
The embedded cationic liposomal gene carrier system of the sucrose ester obtained, different intracellular transfection efficiencies can be poor
It is different.
3, the embedded cationic liposomal gene carrier system of the sucrose ester can enter in tumor-bearing mice cancer cell, complete
The transfection of target gene in the cell.
4, sucrose ester cationic liposomal gene carrier provided by the invention is suitable for coding fluorescence element enzyme, green fluorescence egg
White reporter gene, siRNA needed for being also applied for other various experiments.It is thin that the carrier can efficiently deliver pDNA and siRNA transfection
Born of the same parents.
5, the embedded cationic liposomal gene carrier system of the sucrose ester is nearly free from toxicity to cell and mouse.
The invention has the following advantages over the prior art:
1, the embedded cationic liposomal gene carrier system of sucrose fatty ester provided by the invention, use help lipoid
For sucrose ester.Sucrose ester is a kind of nonionic surfactant, is had extensive hydrophilic lipophilic balance (HLB), is had good
Dispersion, characteristic of solubilizing.Sucrose ester can be enhanced the dispersibility of liposome, enhance lipid as helping lipoid to be used to prepare liposome
The compatibility effect of body and drug accelerates drug releasing rate, promotes dispersion, prevents crystalline deposit, and extends the drug quality guarantee period.
2, the embedded cationic liposomal gene carrier system of sucrose fatty ester provided by the invention, the sucrose of use
Ester has good biocompatibility, good biological degradability, can reduce the toxicity of quaternary liposome.
3, the embedded cationic liposomal gene carrier system of sucrose fatty ester provided by the invention, preparation method letter
Single, the reaction reagent of use and obtained product are nontoxic and pollution-free, and cost of material is low, can be widely applied to scientific research and life
It produces.
4, the embedded cationic liposomal gene carrier system stability of sucrose fatty ester of the present invention is good, grain
Diameter is 200nm or so, and it is good, malicious to improve external transfection efficiency, cell compatibility in vivo for compressible more electronegative genetic stews
Property is small, can be used as the non-viral gene vector and transfection reagent of new and effective low toxicity.
5, the embedded cationic liposomal gene carrier system of sucrose fatty ester provided by the invention, in delivery siRNA
Also there is good effect when for gene silencing, silence efficiency is up to 60%.
6, the embedded cationic liposomal gene carrier system of sucrose fatty ester provided by the invention is used in vivo studies
When, Luciferase gene that can efficiently in silencing tumor-bearing mice lung adenocarcinoma cell, and will not influence the normal growth of mouse, it is right
The liver and kidney of mouse are non-toxic.
Detailed description of the invention
Fig. 1 is sucrose fatty ester embedded cationic-liposome CDO14S, CDO14O, CTA14S, CTA14L, CPA14S
With the droplet measurement figure of CPA14P；
Fig. 2 is sucrose fatty ester embedded cationic-liposome CDO14S, CDO14O, CTA14S, CTA14L, CPA14S
Figure is detected with the Zeta potential of CPA14P；
Fig. 3 be the embedded cationic-liposome CDO14S, CDO14O of sucrose fatty ester, CTA14S, CTA14L,
The electrophoresis retardation experimental result picture of CPA14S, CPA14P and CPA14O and Plasmid DNA；
Fig. 4 be using the embedded cationic-liposome CDO14S, CDO14O of flow cytomery sucrose fatty ester,
CTA14S, CTA14L, CPA14S, CPA14P and CPA14O deliver pGFP-N2 plasmid transfection Hela cell (human cervical carcinoma cell)
Egfp expression amount；
Fig. 5 be using microplate reader detection the embedded cationic-liposome CDO14S, CDO14O of sucrose fatty ester,
CTA14S, CTA14L, CPA14S and CPA14P deliver siRNA and transfect A549 cell, Luciferase gene silencing situation map；
Fig. 6 be using the MTT colorimetric determination embedded cationic-liposome CDO14S of sucrose fatty ester of the invention,
CTA14S and CPA14S and the detection to Hela cytotoxicity in the process of gene composite cell transfecting；
Fig. 7 be using the MTT colorimetric determination embedded cationic-liposome CDO14S of sucrose fatty ester of the invention,
The detection of CTA14S and CPA14S and gene composite to A549 cytotoxicity；
Fig. 8 is sucrose fatty ester embedded cationic-liposome CDO14S, CDO14O, CTA14S, CTA14L, CPA14S
With CPA14P and gene composite cellular uptake situation map；
Fig. 9 is silencing Luciferase in the embedded cationic-liposome CDO14S delivery siRNA body of sucrose fatty ester
Genetic profile and transfection front and back gross tumor volume ratio；
Figure 10 is that mouse is transfected in the embedded cationic-liposome CDO14S of sucrose fatty ester and gene composite body
Changes of weight curve graph.
Specific embodiment
For a further understanding of the present invention, embodiment of the present invention is illustrated below with reference to embodiment, but these
Illustrate only be further explanation the features and advantages of the present invention, rather than limiting to the claimed invention.
In this specification, peptide type cation lipoid indicates that Gemini type cationic lipoid is indicated with CTA14 with CDO14, quaternary ammonium
Salt form cation lipoid indicates that sucrose stearate (HLB < 1) is indicated with S070 with CPA14, and sucrose stearate (HLB=5) is used
S570 indicates that sucrose stearate (HLB=16) indicates that sucrose palmitate (HLB=1) is indicated with P170 with S1670, sucrose palm fibre
Glycerin monostearate (HLB=16) indicates that Surfhope SE Cosme C 1216 (HLB=1) is indicated with L195 with P1670, and sucrose erucate (HLB=2) is used
ER290 indicates that sucrose oleate (HLB=1) is indicated with O170.
The preparation of 1 cationic-liposome CDO14S of embodiment
The peptide type cation lipoid CDO14 of weighing 1mg helps lipoid sucrose stearate S070(1mg with identical mass ratio)
It is dissolved in 1mL methanol and chloroform in the mixed solvent (mixed proportion is 2:1 volume ratio), to after completely dissolution, blow under a nitrogen
At uniform film, being dried in vacuo 5h makes solvent all volatilizations (vacuum degree -0.09MPa, room temperature), molten with 100 μ L dehydrated alcohols
Xie Hou is added 900 μ L ultrapure waters, makes concentration of liposomes 1mg/mL, and in 80 DEG C or so aquation 1h, supersonic frequency is under 100Hz
The embedded cationic-liposome of sucrose ester, concentration 1mL/mg is made to clear in ultrasonic vibration.
The preparation of 2 cationic-liposome CTA14S of embodiment
The Gemini type cationic lipoid CTA14 for weighing 0.5mg is dissolved in 1mL chloroform, and 1mg is added and helps lipoid sucrose stearic
Acid esters S570(CTA14 and S570 mass ratio are 1:2), to after completely dissolution, blow out uniform film, vacuum drying under a nitrogen
12h makes solvent all volatilizations (vacuum degree -0.09MPa, room temperature), impregnates 2h with 1mL ultrapure water, so that film is fallen off, on 55 DEG C of left sides
The embedded sun of sucrose ester that concentration is 0.5mg/mL is made to clear in right ultrasonic vibration (oscillation frequency 100Hz) repeatedly
Cationic liposomal.
The preparation of 3 cationic-liposome CPA14P of embodiment
Weigh 5mg quaternary ammonium salt cationic lipoid CPA14 and 0.625mg help lipoid sucrose palmitate P170(CPA14 and
P-170 mass ratio is 8:1) it is dissolved in 5mL chloroform, to after completely dissolution, blow out uniform film, vacuum drying under a nitrogen
8h makes solvent all volatilizations (vacuum degree -0.09MPa, room temperature), and 5mL phosphate buffer is added, and in 30 DEG C of aquation 8h, surpasses
Acoustic frequency is that for ultrasonic vibration to clear, it is the embedded cationic peptide liposome of 5mg/mL sucrose ester that concentration, which is made, under 100Hz.
The preparation of 4 cationic-liposome CDO14O of embodiment
Precise 2mg peptide type cation lipoid CDO14 and 0.67mg help lipoid sucrose oleate O170(CDO14 with
O170 mass ratio is 3:1), it is dissolved in 1mL methanol and chloroform in the mixed solvent (methanol: chloroform=1:2 volume ratio), to
After completely dissolution, under a nitrogen blow out uniform film, vacuum drying 4h make solvent all volatilization (vacuum degree -0.09MPa, often
Temperature), 55 DEG C or so of 200 μ L of dehydrated alcohol, which is added, makes film fall off, and 800 μ L are added and buffer night, in 40 DEG C or so ultrasound shakes repeatedly
(supersonic frequency 100Hz) is swung to clear, and the embedded cationic-liposome of sucrose ester that concentration is 2mg/mL is made.
The preparation of 5 cationic-liposome CTA14L of embodiment
Precise 2mg peptide type cation lipoid CDO14 and 0.67mg help lipoid sucrose oleate O170(CDO14 with
O170 mass ratio is 3:1), it is dissolved in 1mL methanol and chloroform in the mixed solvent (methanol: chloroform=1:2 volume ratio), to
After completely dissolution, under a nitrogen blow out uniform film, vacuum drying 4h make solvent all volatilization (vacuum degree -0.09MPa, often
Temperature), 55 DEG C or so of 200 μ L of dehydrated alcohol, which is added, makes film fall off, and 800 μ L are added and buffer night, in 40 DEG C or so ultrasound shakes repeatedly
(supersonic frequency 100Hz) is swung to clear, and the embedded cationic-liposome of sucrose ester that concentration is 2mg/mL is made.
The preparation of 6 cationic-liposome CPA14O of embodiment
Precise 1mg quaternary ammonium salt cationic lipoid CPA14 and 1mg help lipoid sucrose oleate O170(CPA14 and O170
Mass ratio is 1:1), it is dissolved in 1mL chloroform solvent, to after completely dissolution, blow out uniform film under a nitrogen, is dried in vacuo 4h
Make solvent all volatilizations (vacuum degree -0.09MPa, room temperature), addition 1mL physiological saline, in 40 DEG C or so ultrasonic vibrations repeatedly
The embedded cationic-liposome of sucrose ester that concentration is 1mg/mL is made to clear in (supersonic frequency 100Hz).
The preparation of 7 cationic-liposome CPA14S of embodiment
Precise 3mg quaternary ammonium salt cationic lipoid CPA14 and 0.5mg help lipoid sucrose stearate S1670(CPA14
It is 6:1 with S1670 mass ratio), it is dissolved in 1mL methanol and chloroform in the mixed solvent (methanol: chloroform=1:2 volume ratio),
To after completely dissolution, blow out uniform film under a nitrogen, vacuum drying 5h make solvent all volatilization (vacuum degree -0.09MPa,
Room temperature), 55 DEG C or so of 200 μ L of physiological saline, which is added, makes film fall off, and 800 μ L ultrapure waters are added, repeatedly ultrasonic at 60 DEG C or so
(supersonic frequency 100Hz) is shaken to clear, and the embedded cationic-liposome of sucrose ester that concentration is 3mg/mL is made.
The partial size and Zeta potential of 8 liposome of embodiment detect
Using laser diffraction particle size instrument (HORIBA nano particle size instrument SZ-100) under the conditions of 25 DEG C, 90 ° of light scattering angle
The partial size and its Zeta potential for measuring the embedded cationic-liposome of sucrose ester of preparation take 20 1 ~ realities of μ L embodiment with liquid-transfering gun
The cationic-liposome for applying the preparation of example 8 is diluted in 1mL ultrapure water carries out partial size and Zeta potential detection, the result is shown in Figure 1 respectively
And Fig. 2.Fig. 1 is sucrose ester embedded cationic-liposome CDO14S, CDO14O, CTA14S, CTA14L, CPA14S and CPA14P
Average grain diameter, Fig. 2 be the embedded cationic-liposome of sucrose ester Zeta potential.
The results show that the embedded cationic-liposome partial size of sucrose ester is between 150 ~ 250nm, in effective grain of transfection
Within the scope of diameter (1 μm of <).Zeta potential absolute value is all larger than 30 mV, has preferable electrostatic stability.
The preparation of 9 CDO14S liposome of embodiment/DNA compound
The embedded cationic-liposome of sucrose ester is prepared in method as described in example 1 above.Take 1mg/mL liposome
0.5 μ g of CDO14S is diluted to 25 μ L with plasma-free DMEM medium；0.5 μ g of 0.5mg/mL Plasmid DNA is taken, with serum-free DMEM
Culture medium is diluted to 25 μ L；Two dilutions mixing (liposome is 1:1 with DNA mass ratio), slight whirlpool concussion, incubation at room temperature
10min obtains CDO14S liposome/DNA compound.
The preparation of 10 CTA14S liposome of embodiment/DNA compound
The embedded cationic-liposome of sucrose ester is prepared in method as described in example 2 above.Take 0.5mg/mL liposome
1 μ g of CTA14S is diluted to 25 μ L with plasma-free DMEM medium；0.5 μ g of 0.5mg/mL Plasmid DNA is taken, with serum-free DMEM
Culture medium is diluted to 25 μ L；Two dilutions mixing (liposome is 2:1 with DNA mass ratio), slight whirlpool concussion, incubation at room temperature
20min obtains CTA14S liposome/DNA compound.
The preparation of 11 CPA14P liposome of embodiment/DNA compound
The embedded cationic-liposome of sucrose ester is prepared in method as described in example 3 above.Take 1.5mg/mL liposome
3.0 μ g of CPA14P is diluted to 25 μ L with plasma-free DMEM medium；0.5 μ g of 0.5mg/mL Plasmid DNA is taken, serum-free is used
DMEM culture medium is diluted to 25 μ L；Two dilutions mixing (liposome is 6:1 with DNA mass ratio), slight whirlpool concussion, room temperature are incubated
30min is educated, CPA14P liposome/DNA compound is obtained.
The preparation of 12 CDO14P liposome of embodiment/DNA compound
Cationic-liposome is prepared in method as described in example 4 above.4.0 μ g of 3.0mg/mL liposome CDO14P is taken,
25 μ L are diluted to plasma-free DMEM medium；0.5 μ g/ μ L Plasmid DNA, 0.5 μ g is taken, is diluted to plasma-free DMEM medium
25µL；Two dilutions mixing (liposome is 8:1 with DNA mass ratio), slight whirlpool concussion are incubated at room temperature 40min, obtain
CDO14P liposome/DNA compound.
The preparation of 13 cationic-liposome CDO14S/siRNA compound of embodiment
Embedded cationic-liposome is prepared in method as described in example 1 above, takes liposome 1mg/mL CDO14S
0.9 μ g is diluted to 25 μ L with plasma-free DMEM medium；0.3 μ g/ μ L siRNA0.3 μ g is taken, plasma-free DMEM medium is used
It is diluted to 25 μ L；Two dilutions mixing (liposome is 3:1 with siRNA mass ratio), slight whirlpool concussion are incubated at room temperature 20min,
Obtain CDO14S liposome/siRNA compound.
The preparation of 14 cationic-liposomes of embodiment/CTA14S/siRNA compound
Embedded cationic-liposome is prepared in method as described in example 2 above, takes liposome 1mg/mL CTA14S
0.5 μ g is diluted to 25 μ L with plasma-free DMEM medium；0.3 μ g/ μ L siRNA, 0.25 μ g is taken, is cultivated with serum-free DMEM
Base is diluted to 25 μ L；Two dilutions mixing (liposome is 2:1 with siRNA mass ratio), slight whirlpool concussion, incubation at room temperature
20min obtains CTA14S/siRNA compound.
The experiment of 15 liposome combination Plasmid DNA electrophoresis retardation of embodiment
It is tested using agarose gel electrophoresis retardation, detects cationic peptide liposome and Plasmid DNA in different quality ratio
Its corresponding charge from which further follows that effective ratio of compression than situation.In mass ratio by cationic peptide liposome and Plasmid DNA
It is followed successively by 0:1,0.5:1,1:1,2:1,3:1,4:1,6:1,8:1 and is diluted in slight whirlpool in 15 μ L RPMI1640 culture solutions respectively
Whirlpool concussion mix, be incubated at room temperature 20 min, take 6 × DNA Loading buffer of 2 μ L sequentially add above-mentioned 15 μ L sun from
It mixes, and is successively splined in 1.2% Ago-Gel loading hole in sub- peptidoliposome and Plasmid DNA compound, voltage is set as
90 V, 40 min of electrophoresis.Nucleic acid dye liquor NA-Red is added when glue, directly in gel imaging system Gene after electrophoresis
DNA retardation situation is observed in Genius Bio-imaging System (SYNGENE company).Electrophoresis result is as shown in Fig. 3,
Wherein swimming lane 1 is Mark, 2 ~ 9 for cationic peptide liposome and DNA be respectively in mass ratio 0:1,0.5:1,1:1,2:1,3:1,
The liposome of 4:1,6:1 and 8:1/DNA compound.Fig. 3 the results show that with lipid weight increase, DNA retardation effect
It is gradually obvious, for liposome CDO14S, CDO14O, CTA14S, CTA14L and CPA14S, be in liposome and DNA mass ratio
DNA complete retardation when 2:1.It is as the result is shown 6:1 in liposome and DNA mass ratio for liposome CPA14P and CPA14O
When DNA can be compressed completely by liposome.
16 extracorporeal biology evaluation experimental of embodiment
(1) delivery pGFP-N2 plasmid-transfected cells experiment
By Hela cell seeding in 24 porocyte culture plates, every hole adds cell concentration to be about 1.0 × 105A/hole is incubated for
After 24 h, make transfecting day cell density up to 80 ~ 90%.By liposome and pGFP-N1 plasmid respectively according to 1:1,2:1,3:1,4:
1,6:1 and 8:1 ratio is compound, and compound rear total volume is 100 μ L.Compound is added in tissue culture plate, cultivates 4-5 h, more
The culture medium containing serum 10% and antibiotic is changed, 48 h are cultivated.Using flow cytomery Green Fluorescent Protein Gene Expression
Amount.It is compareed using commercialization gene transfection agent Lipofectamine 2000.
Experimental result is as shown in Fig. 4, and preferred liposome and DNA mass ratio are 3:1.The embedded liposome base of sucrose ester
Because carrier can deliver pGFP-N1 plasmid transfection Hela cell.With helping lipoid sucrose ester dosage to gradually increase, liposome base
Because carrier transfection efficiency is gradually increased；Wherein, the liposome gene carrier transfection effect that CDO14 and S070 is prepared with mass ratio 1:1
Rate is suitable with commodity transfection reagent.
(2) RNA interference experiment
Plating cells do not count, and take the A549 cell covered with substantially, are added in 12 orifice plates, and every hole adds 2 mL, and culture is about
24 h, cell density are about 50-60%.200 μ L liposomes/siRNA compound is added in every hole, transfects 18 h, changes grown cultures
30 h of base culture.Cell is washed 1 time, 600 μ L lysates of every hole addition with DPBS, after 20 min, is moved in 96 hole blanks, often
Hole adds 20 μ L, and 80 μ L Pu Luomaige E151A is added to detect liquid, detects enzyme activity with multi-function microplate reader (BioTek).It takes
5 μ L of lysate is added in 96 hole transparent panels, using the Pierce BCA Protein Assay kit of power & light company as standard
Blank determination total protein content.
Fig. 5 select liposome and siRNA optimum N/P ratio 3:1, mediate rna i 7 kinds of sucrose esters of study tour it is embedded sun from
The ability of sub- liposomal delivery siRNA transfection A549 cell silencing luciferase gene.Lipofectamine2000 is the positive
Control, siRNA and Control are blank control.After transfecting as the result is shown, the expression of luciferase gene is not by
With the inhibition of degree.Wherein, compared to the blank group, the silence efficiency of luciferase gene is reachable for CDO14O/siRNA genophore
75%.Illustrate that nucleic acid-protein compound (RNA- can be formed with the protein in cytoplasm after liposome-mediated siRNA enters cell
Induced silencing complex, RISC), under the action of RISC, siRNA specific recognition purpose mRNA, in nucleic acid
Enzyme cutting effect is lower to cut purpose mRNA, and mRNA segment can not instruct to synthesize corresponding albumen after Exonucleolytic enzyme effect declines solution
Matter, to realize the silencing to luciferase gene.
(3) Study of cytotoxicity (MTT colorimetric method)
Cell toxicity test is carried out to the higher cationic-liposome of transfection efficiency using mtt assay, is turned so that gene is commercialized
Transfection reagent Lipofectamine 2000 and DOTAP is control.By Hela and A549 cell seeding in 96 porocyte culture plates,
Every hole adds cell culture fluid (containing dual anti-and serum) 100 μ L, and concentration is about 1.0 × 106A/hole is incubated for 24 h, makes transfecting
Day cell density is up to 80 ~ 90 %.Growth medium is removed, is cleaned with 100 μ L culture mediums, then with equivalent (100 μ L) culture medium
Replacement.By liposome and Plasmid DNA respectively according to 1:1,2:1,3:1,4:1,6:1 and 8:1 ratio is compound to be added to tissue culture plate
In.After 24 h of cell culture, 20 μ L MTT(Sigma, 5 mg/mL are added in every hole), it is incubated for culture 4.5 h of 4-.Discard training
The 150 molten broken cells of μ L DMSO are added in nutrient solution.
Testing principle based on MTT colorimetric method are as follows: the succinate dehydrogenase in living cells mitochondria can make exogenous MTT also
Originally it was the bluish violet knot crystalline substance formazan (Formazan) of water-insoluble and was deposited in cell, and dead cell is without this function, dimethyl
Sulfoxide (DMSO) can dissolve the first a ceremonial jade-ladle, used in libation in cell, its light absorption value is measured at 570nm wavelength with microplate reader, can reflect indirectly living thin
Born of the same parents' quantity.So calculating the percentage of Transfected cells survival with the light absorption value of blank control (non-transfected cells) for 100 %
(%).Calculation formula are as follows:
Cell survival rate (%)=[A]Sample/[A]Control×100 %
[A]SampleFor the light absorption value of instrument connection, [A]ControlFor the light absorption value of negative blank control wells.
Experimental result such as Fig. 6 and Fig. 7, abscissa are the prepared embedded cationic-liposome of sucrose ester, preferred rouge
Plastid and DNA mass ratio are 3:1.Fig. 6 is the sucrose ester embedded cationic-liposome base of the invention using MTT colorimetric determination
Because of the detection in carrier transfection process to Hela cytotoxicity.Fig. 7 is the cation lipid of the invention using MTT colorimetric determination
Detection during body genophore cell transfecting to A549 cytotoxicity.The results show that being carried with the increase of sucrose ester dosage
The cytotoxicity of system system is gradually reduced.Under the conditions of best transfection efficiency, poison of the liposome to Hela cell and A549 cell
Property is all little, and cell survival rate is between 80% ~ 100%.
(4) cellular uptake is studied
By A549 cell seeding in 6 porocyte culture plates, every hole adds cell culture fluid (containing dual anti-and serum) 1 mL, dense
Degree about 4.0 × 106A/hole is incubated for 24 h, makes transfecting day cell density up to 70 ~ 80 %.
Liposome and siRNA is compound for 3:1 with mass ratio, and 200 μ L of total volume is added in every hole, cultivates 30 h.With
DPBS is washed once, 0.3 mL trypsin digestion cell of every hole addition, and after 1-2 min, 0.8 mL culture medium is added.It, will be thin after centrifugation
Born of the same parents are diluted with 0.4 mL DPBS, use flow cytometry analysis.
Fig. 8 is cellular uptake testing result, and the embedded cationic-liposome of sucrose ester and siRNA mass ratio are 3:1.6 kinds
Liposome can enter cell, and cellular uptake rate is between 30 ~ 80%.Wherein, liposome CDO14S(CDO14 and S070 mass ratio
Reach 76 % for 1:1) cellular uptake amount.
17 vivo biodistribution evaluation experimental of embodiment
(1) foundation of mouse model
Logarithmic growth phase Luc-A549 cell, is washed once with DPBS, adds 3 mL trypsin digestions, 5 mL is added to have blood
Cleer and peaceful dual anti-PRMI1640, gently blows and beats, and is transferred in 15 mL centrifuge tubes, 5 min are centrifuged at 1200 rpm.In removal
Clear liquid is diluted in 1.5 mL PBS, concentration is made to reach 2 × 107cells/mL.After dropping nude mice disinfection, plant in nude mice forelimb armpit
Lower subcutaneous, every about 200 μ L, tumour grows to 100-200 mm3When, it can be used.
(2) siRNA silencing Luciferase gene is delivered
12 tumor-bearing mices are randomly divided into 4 groups, every group 3, respectively blank control group, CDO14S liposome transfection
Group, positive controls (Lipofectamine2000 transfection group).Liposome/siRNA is compound with mass ratio 3:1, and dosage is
8 μ g/20g of siRNA, 24 μ g/20g of liposome.Compound through tail vein injection in tumor-bearing mice, before transfection and after transfection
For 24 hours, 48h, 72h measure and record nude mice weight, survey tumor size, and calculate knurl product.Living imaging detects internal luciferase
Expression, nude mice abdominal cavity injected fluorescein 3mg/20g move freely after 15min with halothane anesthesia, enters back into living imaging system
It takes pictures.To transfect effect of the net optical density than indicating internal RNAi in the mouse tumor of front and back.
Cationic-liposome R18ORRR delivers siRNA, the result of silencing luciferase gene in mouse tumor cell
As shown in Fig. 9, after injecting lipid body CDO14S for 24 hours, mouse tumor volume becomes larger, but in mouse tumor cell
Luciferase fluorescence intensity reduces by 50%.Illustrate that the embedded cationic-liposome CDO14S of sucrose ester can specifically, efficiently
Lower the overexpression of luciferase gene.
(3) in vivo cytotoxicity is studied
Liposome/siRNA is compound with mass ratio 3:1, through tail vein injection in tumor-bearing mice, once every injection in two days,
Co-injection 5 times.Mouse weight is measured before daily administration, observes the variation of the daily body weight increase of each group mouse.Mouse is measured simultaneously
Biochemical Indices In Serum investigates the embedded cationic-liposome of sucrose ester to the toxicity of mouse liver and kidney.
The changes of weight of mouse is as shown in Figure 10, and before injection complex, the weight of mouse is 21-23g, injection complex
Mouse weight reaches 25-28 g, shows mouse to certain agent in the trend that rises overally, the average weight of the 9th day mouse in 9 days afterwards
The adaptation of the liposome CDO14S of amount, liposome will not influence the normal growth of mouse.Table 1 is that mice serum biochemistry refers to after transfecting
Mark, the creatinine, urea nitrogen, glutamic-oxalacetic transaminease of mouse and glutamic-pyruvic transaminase changes of contents are little after transfection, in range of normal value,
Illustrate that the embedded cationic-liposome of sucrose ester of the invention does not generate toxicity to the liver and kidney of mouse.
Table 1 is the embedded cationic-liposome CDO14S of sucrose fatty ester and gene composite to mouse liver and kidney
Toxicity.
Table 1
。
Claims (4)
1. a kind of embedded cationic liposomal gene carrier system of sucrose fatty ester, it is characterised in that: the Sucrose Fatty Acid Ester
The embedded cationic liposomal gene carrier system of fat acid esters is by the embedded cationic-liposome of sucrose fatty ester and gene object
Matter composition, the embedded cationic-liposome of the sucrose fatty ester are made of cation lipoid and sucrose fatty ester,
The sucrose fatty ester is monoesters or dibasic acid esters, and wherein monoesters structure is as follows:
,
Wherein, R is selected from stearic acid or lauric acid or oleic acid or palmitinic acid or nutmeg acid or erucic acid；
The hydrophilic lipophilic balance of the sucrose fatty ester are as follows: HLB < 1 or 1≤HLB≤16.
2. the embedded cationic liposomal gene carrier system of sucrose fatty ester according to claim 1, feature exist
In: the cation lipoid is peptide type cation lipoid, structural formula are as follows:
；
Or Gemini type cationic lipoid, structural formula are as follows:
；
Or quaternary ammonium salt cationic lipoid, structural formula are as follows:
；
Wherein: x is selected from 1~6；Y is selected from 1~8；R is selected from C8-20 Alkyl, the alkyl include straight chained alkyl and branched alkyl；AA choosing
From arginine or histidine or aspartic acid or alanine or glycine or ornithine or lysine；B is selected from Orn or Lys.
3. the embedded cationic liposomal gene carrier system of sucrose fatty ester according to claim 2, feature exist
In: the mass ratio of the cation lipoid and sucrose fatty ester is 1 ︰, 8~8 ︰ 1；The embedded sun of sucrose fatty ester
The mass ratio of cationic liposomal and genetic stew is 0.5 ︰, 1 ~ 32 ︰ 1.
4. the embedded cationic liposomal gene carrier system of sucrose fatty ester according to claim 1 or 2 or 3,
Be characterized in that: the genetic stew is Plasmid DNA or siRNA.
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