CN105521497A - A sucrose fatty acid ester embedded type cationic liposome genetic vector system, a preparing method thereof and applications of the method - Google Patents

A sucrose fatty acid ester embedded type cationic liposome genetic vector system, a preparing method thereof and applications of the method Download PDF

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CN105521497A
CN105521497A CN201511011257.8A CN201511011257A CN105521497A CN 105521497 A CN105521497 A CN 105521497A CN 201511011257 A CN201511011257 A CN 201511011257A CN 105521497 A CN105521497 A CN 105521497A
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fatty acid
acid ester
sucrose fatty
liposome
cationic
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CN105521497B (en
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张树彪
赵轶男
崔诗慧
赫泽坤
徐宇虹
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Dalian Minzu University
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Dalian Nationalities University
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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
    • A61K47/30—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
    • 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
    • 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
    • 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
    • A61K48/0033—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
    • 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
    • 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 invention relates to a sucrose fatty acid ester embedded type cationic liposome genetic vector system, a preparing method thereof and applications of the method. The genetic vector system is a composition comprising genetic materials and liposomes formed from cationic lipoids and sucrose esters adopted as auxiliary lipoids. The cationic lipoids comprise peptide type cationic lipoids, gemini cationic lipoids and quaternary ammonium salt type cationic lipoids. The sucrose esters comprise sucrose stearate, sucrose palmitate, sucrose laurate, sucrose oleate and sucrose erucate. The vector can effectively compress plasmid DNA and siRNA and is capable of efficient transfection in vitro and in vivo and nearly free of toxicity for cells and mice. The genetic vector system has a good research and application prospect in gene therapy.

Description

Embedded cationic liposomal gene carrier system of sucrose fatty acid ester and its preparation method and application
Technical field
The invention belongs to biological technical field, particularly embedded cationic liposomal gene carrier system of sucrose fatty acid ester and preparation method thereof.
Technical background
Gene therapy (genetherapy) refers to and external source normal gene is imported target cell, to correct or to compensate because of genetic flaw and the abnormal disease caused, to reach therapeutic purposes.Nineteen ninety, NIH adopted gene therapy method to treat adenosine deaminase deficiency disease, is applied to clinical first and achieves gene therapy successfully.Within 2000, adopt gene therapy method successful treatment combined immunodeficiency syndrome, indicate the effectiveness of strategies in gene therapy.Gene therapy will further be developed, and must overcome a difficult problem for three aspects: one, will have the genes of interest be used for the treatment of and accept the cell of genes of interest; Two, the Modulatory character of gene expression is realized; Three, the efficient Gene delivery system with targeting is obtained.Wherein, have the efficient Gene delivery system of targeting, namely genophore becomes the key factor of restriction gene therapy development.
Genophore mainly contains two large classes: viral genetic vector, non-viral gene vector.Since the clinical trial being successfully made gene therapy first, major part adopts the high viral vector of transfection efficiency, but viral vector has very high immunogenicity, is easy to recombinate with wild-type virus, and carrying capacity is limited, production cost is high and easily contaminated.University of Pennsylvania of the U.S. in 1999 is that the gene therapy medicament of carrier causes 18 years old patients die in clinical trial with recombinant virus.Within 2000, French Nei Keer hospital applies retroviral vector in the process for the treatment of severe combined immunodeficiency, finds that there is several children create leukemia in 2003.Therefore the development of viral vector is restricted.Non-viral gene vector is compared with viral genetic vector, although transfection efficiency is relatively low, but there is reduced immunogenicity, low toxicity, foreign DNA intergration rate are low, nucleus or chromosome can be delivered without the restriction of gene insert size, even, use is simple, the advantage such as easy to prepare.Increasing research worker starts to select non-virus carrier to carry out the research of gene therapy aspect.Cationic-liposome is at present research the most thorough, the most ripe, the most widely used non-viral carrier systems of technology, has class cellularity and biomembranous characteristic, in vivo degradable, can protect the biological activity of its passenger gene fragment.But it also has some limitations, as cytotoxicity is large, not obvious to the targeting of organ, gene delivery mechanism is indefinite, limits the development of cationic-liposome.Therefore, people are devoted to various structure to cationic-liposome and modification always, attempt the gene therapy medicament seeking a kind of high-efficiency low-toxicity.
Sucrose ester is a kind of non-ionic surface active agent, the simple substance generated through esterification by sucrose and fatty acid or mixture.Sucrose ester has hydrophile-lipophile balance value (HLB) widely, has good dispersion, characteristic of solubilizing.Therefore, sucrose ester can strengthen the dispersibility of liposome as helping lipoid for the preparation of liposome, strengthens the compatibility effect of liposome and medicine, accelerates drug releasing rate, promotes dispersion, prevents crystalline deposit, and the prolong drug quality guarantee period.In addition, sucrose ester biocompatibility, biological degradability are strong, can reduce the toxicity of liposome for the preparation of cationic-liposome.
Summary of the invention
The object of this invention is to provide embedded cationic liposomal gene carrier system of little, that efficiency gene transfection the is high in vitro and in vivo sucrose fatty acid ester of a kind of cytotoxicity and preparation method thereof and application thereof, overcome the deficiencies in the prior art.
Sucrose fatty acid ester of the present invention embedded cationic liposomal gene carrier system is made up of the embedded cationic-liposome of sucrose fatty acid ester and genetic stew, and the embedded cationic-liposome of described sucrose fatty acid ester is made up of cation lipoid and sucrose fatty acid ester.
The embedded cationic liposomal gene carrier system of sucrose fatty acid ester of the present invention, wherein said sucrose fatty acid 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 Palmic acid or Semen Myristicae acid or erucic acid;
The hydrophile-lipophile balance value of described sucrose fatty acid ester is: HLB<1 or 1≤HLB≤16.
The embedded cationic liposomal gene carrier system of sucrose fatty acid ester of the present invention, wherein said cation lipoid is peptide type cation lipoid, and structural formula is:
；
Or Gemini type cationic lipoid, structural formula is:
；
Or quaternary ammonium salt cationic lipoid, structural formula is:
；
Wherein: x is selected from 1 ~ 6; Y is selected from 1 ~ 8; R is selected from C 8-20alkyl, this alkyl comprises 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 acid ester of the present invention, wherein said cation lipoid and the mass ratio of sucrose fatty acid ester are 1 ︰ 8 ~ 8 ︰ 1; The mass ratio of the embedded cationic-liposome of described sucrose fatty acid ester and genetic stew is 0.5 ︰ 1 ~ 32 ︰ 1.
The embedded cationic liposomal gene carrier system of sucrose fatty acid ester of the present invention, wherein said genetic stew is plasmid DNA or siRNA.
The preparation method of the embedded cationic liposomal gene carrier system of sucrose fatty acid ester of the present invention, step is as follows:
The preparation of the embedded cationic-liposome of sucrose fatty acid ester:
(1) cation lipoid and sucrose fatty acid ester to be dissolved in chloroform or methanol to obtain solution according to mass ratio 1:8 ~ 8:1 ratio;
(2) solution (1) step obtained blows out homogeneous film under a nitrogen, vacuum drying 2 ~ 12h;
(3) carry out aquation with ethanol or water or normal saline or phosphoric acid as buffer or with the mixed liquor of the quality such as any two kinds in ethanol, water, normal saline, phosphoric acid, hydration temperature is 10 ~ 80 DEG C; Hydration time is 1 ~ 10h, and ultrasonic vibration is to clarification, and obtain the embedded cationic-liposome of sucrose fatty acid ester, concentration is 0.5 ~ 3.0mg/mL; The embedded cationic-liposome of this sucrose fatty acid ester is be dispersed in the cationic-liposome of surface with positive charge that the size formed in aqueous phase is the stable homogeneous of 150 ~ 250nm;
The embedded cationic-liposome of sucrose fatty acid ester and genetic stew is utilized to prepare the embedded cationic liposomal gene carrier system of sucrose fatty acid ester:
By embedded for sucrose fatty acid ester cationic-liposome and plasmid DNA or siRNA, formed the nano-particle being dispersed in stable homogeneous in aqueous phase by electrostatic interaction, concrete steps are as follows:
(1) getting the embedded cationic-liposome of sucrose fatty acid ester is dispersed in cell culture fluid DMEM or RPMI1640, and mixing, makes concentration be 0.01 μ g/ μ L ~ 0.64 μ g/ μ L;
(2) be diluted in cell culture fluid DMEM or RPMI1640 by 0.5 ~ 1.0 μ g plasmid DNA or siRNA, mixing, makes plasmid concentration be 0.02 μ g/ μ L;
(3) according to the embedded cationic-liposome of sucrose fatty acid ester and genetic stew mass ratio 0.5:1 ~ 32:1 ratio, by (1) and the mixing of (2) two diluents, room temperature places 10 ~ 40min, can obtain the embedded cationic liposomal gene carrier system of sucrose fatty acid ester.
Application in the application of the embedded cationic liposomal gene carrier system of sucrose fatty acid ester prepared of the preparation method of sucrose fatty acid ester of the present invention embedded cationic liposomal gene carrier system in cell transfecting and in vivo transfection, mainly comprises:
1, the embedded cationic liposomal gene carrier of described sucrose ester can enter in cancerous cell, completes genes of interest in intracellular transfection.
2, described cell is human cervical carcinoma cell (Hela) and non-little lung adenocarcinoma cell (A549).In conjunction with the embedded cationic liposomal gene carrier system of described sucrose ester of acquisition under different N/P ratio, can difference to some extent at the intracellular transfection efficiency of difference.
3, the embedded cationic liposomal gene carrier system of described sucrose ester can enter in tumor-bearing mice cancerous cell, completes genes of interest in intracellular transfection.
4, sucrose ester cationic liposomal gene carrier provided by the invention is applicable to coding fluorescence element enzyme, Green fluorescent protein fusion vector, is also applicable to other siRNA needed for various experiment.This carrier can efficiently deliver pDNA and siRNA transfectional cell.
5, the embedded cationic liposomal gene carrier system of described sucrose ester produces toxicity hardly to cell and mice.
The present invention compared with prior art tool has the following advantages:
1, the embedded cationic liposomal gene carrier system of sucrose fatty acid ester provided by the invention, the lipoid that helps of employing is sucrose ester.Sucrose ester is a kind of non-ionic surface active agent, has hydrophile-lipophile balance value (HLB) widely, has good dispersion, characteristic of solubilizing.Sucrose ester can strengthen the dispersibility of liposome as helping lipoid for the preparation of liposome, strengthens the compatibility effect of liposome and medicine, accelerates drug releasing rate, promotes dispersion, prevents crystalline deposit, and the prolong drug quality guarantee period.
2, the embedded cationic liposomal gene carrier system of sucrose fatty acid ester provided by the invention, the sucrose ester of employing has good biocompatibility, good biological degradability, can reduce the toxicity of quaternary liposome.
3, the embedded cationic liposomal gene carrier system of sucrose fatty acid ester provided by the invention, preparation method is simple, the reaction reagent of employing and the product nontoxic pollution-free obtained, and cost of material is low, can be widely used in scientific research and production.
4, sucrose fatty acid ester of the present invention embedded cationic liposomal gene carrier system good stability, particle diameter is about 200nm, compressible more how electronegative genetic stew, improve transfection efficiency in external body, cell compatibility is good, toxicity is little, can be used as non-viral gene vector and the transfection reagent of new and effective low toxicity.
5, the embedded cationic liposomal gene carrier system of sucrose fatty acid ester provided by the invention, also have good effect when delivering siRNA and being used for gene silencing, its silence efficiency is up to 60%.
6, the embedded cationic liposomal gene carrier system of sucrose fatty acid ester provided by the invention, during in vivo test, can Luciferase gene in efficient reticent tumor-bearing mice lung adenocarcinoma cell, and the normal growth of mice can not be affected, to liver and the kidney avirulence of mice.
Accompanying drawing explanation
Fig. 1 is the droplet measurement figure of sucrose fatty acid ester embedded cationic-liposome CDO14S, CDO14O, CTA14S, CTA14L, CPA14S and CPA14P;
Fig. 2 is the Zeta potential detection figure of sucrose fatty acid ester embedded cationic-liposome CDO14S, CDO14O, CTA14S, CTA14L, CPA14S and CPA14P;
Fig. 3 is the electrophoresis retardation experimental result picture of sucrose fatty acid ester embedded cationic-liposome CDO14S, CDO14O, CTA14S, CTA14L, CPA14S, CPA14P and CPA14O and plasmid DNA;
Fig. 4 is the egfp expression amount adopting flow cytomery sucrose fatty acid ester embedded cationic-liposome CDO14S, CDO14O, CTA14S, CTA14L, CPA14S, CPA14P and CPA14O to deliver pGFP-N2 plasmid transfection Hela cell (human cervical carcinoma cell);
Fig. 5 is that sucrose fatty acid ester embedded cationic-liposome CDO14S, CDO14O, CTA14S, CTA14L, CPA14S and the CPA14P adopting microplate reader to detect delivers siRNA transfection A549 cell, Luciferase gene silencing situation map;
Fig. 6 adopts in MTT colorimetric determination sucrose fatty acid ester of the present invention embedded cationic-liposome CDO14S, CTA14S and CPA14S and gene composite cell transfecting process the Cytotoxic detection of Hela;
Fig. 7 adopts MTT colorimetric determination sucrose fatty acid ester of the present invention embedded cationic-liposome CDO14S, CTA14S and CPA14S and gene composite to the Cytotoxic detection of A549;
Fig. 8 is sucrose fatty acid ester embedded cationic-liposome CDO14S, CDO14O, CTA14S, CTA14L, CPA14S and CPA14P and gene composite cellular uptake situation map;
Fig. 9 is that sucrose fatty acid ester embedded cationic-liposome CDO14S to deliver in siRNA body gross tumor volume ratio before and after reticent Luciferase genetic profile and transfection;
Figure 10 is the body weight change curve chart of transfected in the embedded cationic-liposome CDO14S of sucrose fatty acid ester and gene composite body.
Detailed description of the invention
In order to understand the present invention further, below in conjunction with embodiment, embodiment of the present invention is set forth, but these are set forth just for further illustrating the features and advantages of the present invention, instead of limiting to the claimed invention.
In this description, peptide type cation lipoid CDO14 represents, Gemini type cationic lipoid CTA14 represents, quaternary ammonium salt cationic lipoid CPA14 represents, sucrose stearate (HLB<1) represents with S070, sucrose stearate (HLB=5) represents with S570, sucrose stearate (HLB=16) represents with S1670, sucrose palmitate (HLB=1) represents with P170, sucrose palmitate (HLB=16) represents with P1670, Surfhope SE Cosme C 1216 (HLB=1) represents with L195, sucrose erucate (HLB=2) represents with ER290, sucrose oleate (HLB=1) represents with O170.
The preparation of embodiment 1 cationic-liposome CDO14S
What weigh peptide type cation lipoid CDO14 and the equal in quality ratio of 1mg helps lipoid sucrose stearate S070(1mg) be dissolved in (mixed proportion is 2:1 volume ratio) in 1mL methanol and chloroform mixed solvent, after fully dissolving, blow out homogeneous film under a nitrogen, vacuum drying 5h makes solvent all volatilize, and (vacuum is-0.09MPa, room temperature), after 100 μ L anhydrous alcohol solutions, add 900 μ L ultra-pure waters, concentration of liposomes is made to be 1mg/mL, at about 80 DEG C aquation 1h, supersonic frequency be under 100Hz ultrasonic vibration to clear, the obtained embedded cationic-liposome of sucrose ester, concentration is 1mL/mg.
The preparation of embodiment 2 cationic-liposome CTA14S
The Gemini type cationic lipoid CTA14 weighing 0.5mg is dissolved in 1mL chloroform, adding 1mg helps lipoid sucrose stearate S570(CTA14 and S570 mass ratio to be 1:2), after fully dissolving, blow out homogeneous film under a nitrogen, vacuum drying 12h makes solvent all volatilize, and (vacuum is-0.09MPa, room temperature), 2h is soaked with 1mL ultra-pure water, film is come off, at about 55 DEG C, ultrasonic vibration (concussion frequency is 100Hz) is to clear repeatedly, and obtained concentration is the embedded cationic-liposome of sucrose ester of 0.5mg/mL.
The preparation of embodiment 3 cationic-liposome CPA14P
Quaternary ammonium salt cationic lipoid CPA14 and the 0.625mg weighing 5mg helps lipoid sucrose palmitate P170(CPA14 and P-170 mass ratio to be 8:1) be dissolved in 5mL chloroform, after fully dissolving, blow out homogeneous film under a nitrogen, vacuum drying 8h makes solvent all volatilize, and (vacuum is-0.09MPa, room temperature), add 5mL phosphate buffer, at 30 DEG C of aquation 8h, supersonic frequency is that under 100Hz, ultrasonic vibration is to clear, and obtained concentration is the embedded cationic peptide liposome of 5mg/mL sucrose ester.
The preparation of embodiment 4 cationic-liposome CDO14O
Precise 2mg peptide type cation lipoid CDO14 and 0.67mg helps lipoid sucrose oleate O170(CDO14 and O170 mass ratio to be 3:1), be dissolved in (methanol: chloroform=1:2 volume ratio) in 1mL methanol and chloroform mixed solvent, after fully dissolving, blow out homogeneous film under a nitrogen, vacuum drying 4h makes solvent all volatilize, and (vacuum is-0.09MPa, room temperature), the dehydrated alcohol adding about 200 μ L55 DEG C makes film come off, add 800 μ L and cushion night, about 40 DEG C repeatedly ultrasonic vibration (supersonic frequency is 100Hz) to clear, obtained concentration is the embedded cationic-liposome of sucrose ester of 2mg/mL.
The preparation of embodiment 5 cationic-liposome CTA14L
Precise 2mg peptide type cation lipoid CDO14 and 0.67mg helps lipoid sucrose oleate O170(CDO14 and O170 mass ratio to be 3:1), be dissolved in (methanol: chloroform=1:2 volume ratio) in 1mL methanol and chloroform mixed solvent, after fully dissolving, blow out homogeneous film under a nitrogen, vacuum drying 4h makes solvent all volatilize, and (vacuum is-0.09MPa, room temperature), the dehydrated alcohol adding about 200 μ L55 DEG C makes film come off, add 800 μ L and cushion night, about 40 DEG C repeatedly ultrasonic vibration (supersonic frequency is 100Hz) to clear, obtained concentration is the embedded cationic-liposome of sucrose ester of 2mg/mL.
The preparation of embodiment 6 cationic-liposome CPA14O
Precise 1mg quaternary ammonium salt cationic lipoid CPA14 and 1mg helps lipoid sucrose oleate O170(CPA14 and O170 mass ratio to be 1:1), be dissolved in 1mL chloroform solvent, after fully dissolving, blow out homogeneous film under a nitrogen, vacuum drying 4h makes solvent all volatilize, and (vacuum is-0.09MPa, room temperature), add 1mL normal saline, at about 40 DEG C, ultrasonic vibration (supersonic frequency is 100Hz) is to clear repeatedly, and obtained concentration is the embedded cationic-liposome of sucrose ester of 1mg/mL.
The preparation of embodiment 7 cationic-liposome CPA14S
Precise 3mg quaternary ammonium salt cationic lipoid CPA14 and 0.5mg helps lipoid sucrose stearate S1670(CPA14 and S1670 mass ratio to be 6:1), be dissolved in (methanol: chloroform=1:2 volume ratio) in 1mL methanol and chloroform mixed solvent, after fully dissolving, blow out homogeneous film under a nitrogen, vacuum drying 5h makes solvent all volatilize, and (vacuum is-0.09MPa, room temperature), the normal saline adding about 200 μ L55 DEG C makes film come off, add 800 μ L ultra-pure waters, about 60 DEG C repeatedly ultrasonic vibration (supersonic frequency is 100Hz) to clear, obtained concentration is the embedded cationic-liposome of sucrose ester of 3mg/mL.
The particle diameter of embodiment 8 liposome and Zeta potential detect
Adopt laser diffraction particle size instrument (HORIBA nano particle size instrument SZ-100) at 25 DEG C, particle diameter and the Zeta potential thereof of the embedded cationic-liposome of sucrose ester of preparation is measured under light scattering angle 90 ° of conditions, be diluted in 1mL ultra-pure water with the cationic-liposome that liquid-transfering gun gets 20 μ L embodiment 1 ~ embodiments 8 preparations and carry out particle diameter and Zeta potential respectively and detect, the results are shown in Figure 1 and Fig. 2.Fig. 1 is the mean diameter of sucrose ester embedded cationic-liposome CDO14S, CDO14O, CTA14S, CTA14L, CPA14S and CPA14P, and Fig. 2 is the Zeta potential of the embedded cationic-liposome of sucrose ester.
Result show, the embedded cationic-liposome particle diameter of sucrose ester between 150 ~ 250nm, within effective grain size (the < 1 μm) scope of transfection.Zeta potential absolute value is all greater than 30mV, has good electrostatic stability.
The preparation of embodiment 9CDO14S liposome/DNA complex
Method prepares the embedded cationic-liposome of sucrose ester as described in example 1 above.Get 1mg/mL liposome CDO14S0.5 μ g plasma-free DMEM medium and be diluted to 25 μ L; Get 0.5mg/mL plasmid DNA 0.5 μ g, be diluted to 25 μ L with plasma-free DMEM medium; Two diluents mixing (liposome and DNA mass ratio are 1:1), slight whirlpool concussion, incubated at room 10min, obtains CDO14S liposome/DNA complex.
The preparation of embodiment 10CTA14S liposome/DNA complex
Method prepares the embedded cationic-liposome of sucrose ester as described in example 2 above.Get 0.5mg/mL liposome CTA14S1 μ g, be diluted to 25 μ L with plasma-free DMEM medium; Get 0.5mg/mL plasmid DNA 0.5 μ g, be diluted to 25 μ L with plasma-free DMEM medium; Two diluents mixing (liposome and DNA mass ratio are 2:1), slight whirlpool concussion, incubated at room 20min, obtains CTA14S liposome/DNA complex.
The preparation of embodiment 11CPA14P liposome/DNA complex
Method prepares the embedded cationic-liposome of sucrose ester as described in example 3 above.Get 1.5mg/mL liposome CPA14P3.0 μ g, be diluted to 25 μ L with plasma-free DMEM medium; Get 0.5mg/mL plasmid DNA 0.5 μ g, be diluted to 25 μ L with plasma-free DMEM medium; Two diluents mixing (liposome and DNA mass ratio are 6:1), slight whirlpool concussion, incubated at room 30min, obtains CPA14P liposome/DNA complex.
The preparation of embodiment 12CDO14P liposome/DNA complex
Method prepares cationic-liposome as described in example 4 above.Get 3.0mg/mL liposome CDO14P4.0 μ g, be diluted to 25 μ L with plasma-free DMEM medium; Get 0.5 μ g/ μ L plasmid DNA 0.5 μ g, be diluted to 25 μ L with plasma-free DMEM medium; Two diluents mixing (liposome and DNA mass ratio are 8:1), slight whirlpool concussion, incubated at room 40min, obtains CDO14P liposome/DNA complex.
The preparation of embodiment 13 cationic-liposome CDO14S/siRNA complex
Method prepares embedded cationic-liposome as described in example 1 above, gets liposome 1mg/mLCDO14S0.9 μ g, is diluted to 25 μ L with plasma-free DMEM medium; Get 0.3 μ g/ μ LsiRNA0.3 μ g, be diluted to 25 μ L with plasma-free DMEM medium; Two diluents mixing (liposome and siRNA mass ratio are 3:1), slight whirlpool concussion, incubated at room 20min, obtains CDO14S liposome/siRNA complex.
The preparation of embodiment 14 cationic-liposomes/CTA14S/siRNA complex
Method prepares embedded cationic-liposome as described in example 2 above, gets liposome 1mg/mLCTA14S0.5 μ g, is diluted to 25 μ L with plasma-free DMEM medium; Get 0.3 μ g/ μ LsiRNA0.25 μ g, be diluted to 25 μ L with plasma-free DMEM medium; Two diluents mixing (liposome and siRNA mass ratio are 2:1), slight whirlpool concussion, incubated at room 20min, obtains CTA14S/siRNA complex.
Embodiment 15 liposome is tested in conjunction with plasmid DNA electrophoresis retardation
Adopt the experiment of agarose gel electrophoresis retardation, detect cationic peptide liposome and plasmid DNA different quality than time its corresponding charge ratio situation, draw effective ratio of compression further.Cationic peptide liposome and plasmid DNA are followed successively by mass ratio 0:1,0.5:1,1:1,2:1,3:1,4:1,6:1,8:1 to be diluted in slight whirlpool in 15 μ LRPMI1640 culture fluid respectively and to shake and mix, incubated at room 20min, 6 × the DNALoadingbuffer getting 2 μ L adds successively in above-mentioned 15 μ L cationic peptide liposomees and plasmid dna complex compound and mixes, and be splined in the agarose gel loading hole of 1.2% successively, voltage is set to 90V, electrophoresis 40min.Added nucleic acid dye liquor NA-Red during glue, electrophoresis terminates rear directly observation DNA retardation situation in gel imaging system GeneGeniusBio-imagingSystem (SYNGENE company).As shown in Figure 3, wherein swimming lane 1 is Mark to electrophoresis result, and 2 ~ 9 is liposome/DNA complex that cationic peptide liposome and DNA are respectively 0:1,0.5:1,1:1,2:1,3:1,4:1,6:1 and 8:1 in mass ratio.Fig. 3 result shows, and along with the increase of liposome quality, DNA retardation effect is also obvious gradually, for liposome CDO14S, CDO14O, CTA14S, CTA14L and CPA14S, and the DNA retardation completely when liposome and DNA mass ratio are 2:1.For liposome CPA14P and CPA14O, result is presented at DNA when liposome and DNA mass ratio are 6:1 and can be compressed by liposome completely.
Embodiment 16 extracorporeal biology evaluation experimental
(1) experiment of pGFP-N2 plasmid-transfected cells is delivered
By Hela cell seeding in 24 porocyte culture plates, every hole adds cell concentration and is about 1.0 × 10 5individual/hole, after hatching 24h, make transfection day cell density reach 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 compound, after compound, cumulative volume is 100 μ L.Complex is added in Tissue Culture Plate, cultivates 4-5h, change containing serum 10% and antibiotic culture medium, cultivate 48h.Adopt flow cytomery Green Fluorescent Protein Gene Expression amount.Commercialization gene transfection agent Lipofectamine2000 is adopted to contrast.
As shown in Figure 4, preferred liposome and DNA mass ratio are 3:1 to experimental result.The embedded liposome gene carrier of sucrose ester all can deliver pGFP-N1 plasmid transfection Hela cell.Along with helping lipoid sucrose ester dosage to increase gradually, liposome gene carrier transfection efficiency increases gradually; Wherein, the liposome gene carrier transfection efficiency prepared with mass ratio 1:1 of CDO14 and S070 is suitable with commodity transfection reagent.
(2) RNA interference experiment
Plating cells does not count, and gets the A549 cell substantially covered with, and be added in 12 orifice plates, every hole adds 2mL, and cultivate about 24h, cell density is about 50-60%.Every hole adds 200 μ L liposome/siRNA complex, transfection 18h, changes growth medium and cultivates 30h.Cell DPBS is washed 1 time, and every hole adds 600 μ L lysates, and after 20min, move in 96 hole blanks, every hole adds 20 μ L, adds 80 μ L Pu Luomaige E151A and detects liquid, detect enzyme activity by multi-functional microplate reader (BioTek).Getting lysate 5 μ L is added in 96 hole lamella lucidas, with the PierceBCAProteinAssay test kit of power & light company for standard control measures total protein content.
Fig. 5 selects liposome and siRNA optimum N/P than 3:1, the ability of the reticent luciferase gene of mediate rna i study tour 7 kinds of sucrose ester embedded cationic-liposome delivery siRNA transfection A549 cell.Lipofectamine2000 is positive control, siRNA and Control is blank.After result display transfection, the expression of luciferase gene is all subject to suppression in various degree.Wherein, CDO14O/siRNA genophore is compared with blank group, and the silence efficiency of luciferase gene can reach 75%.Illustrate that liposome-mediated siRNA can form nucleic acid-protein complex (RNA-inducedsilencingcomplex with the protein in Cytoplasm after entering cell, RISC), under the effect of RISC, siRNA specific recognition object mRNA, object mRNA is cut under Cobra venom endonuclease effect, mRNA fragment cannot instruct the corresponding protein of synthesis after degrading under exonuclease effect, thus achieves the silence to luciferase gene.
(3) Study of cytotoxicity (MTT colorimetry)
The mtt assay cationic-liposome higher to transfection efficiency is adopted to carry out cell toxicity test, with commercialization gene transfection agent Lipofectamine2000 and DOTAP for contrast.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 × 10 6individual/hole, hatches 24h, make transfection day cell density reach 80 ~ 90%.Remove growth medium, with 100 μ L culture medium cleanings, then use equivalent (100 μ L) culture medium to replace.Liposome and plasmid DNA are added in Tissue Culture Plate according to 1:1,2:1,3:1,4:1,6:1 and 8:1 ratio compound respectively.After cell culture 24h, in every hole, add 20 μ LMTT(Sigma, 5mg/mL), hatch and cultivate 4-4.5h.Discard culture fluid, add the molten broken cell of 150 μ LDMSO.
Cleaning Principle based on MTT colorimetry is: the succinate dehydrogenase in living cells mitochondrion can make exogenous MTT be reduced to water-insoluble bluish violet Jie Jing formazan (Formazan) and be deposited in cell, and dead cell is without this function, first a ceremonial jade-ladle, used in libation in dimethyl sulfoxide (DMSO) energy dissolved cell, measure its light absorption value by microplate reader at 570nm wavelength place, can indirectly reflect living cells quantity.So, with the light absorption value of blank (non-transfected cells) for 100%, calculate the percentage rate (%) of Transfected cells survival.Computing formula is:
Cell survival rate (%)=[A] sample/ [A] contrast× 100%
[A] samplefor the light absorption value of instrument connection, [A] contrastfor the light absorption value of negative blank control wells.
Experimental result is as Fig. 6 and Fig. 7, and abscissa is the prepared embedded cationic-liposome of sucrose ester, and preferred liposome and DNA mass ratio are 3:1.Fig. 6 adopts in MTT colorimetric determination sucrose ester of the present invention embedded cationic liposomal gene carrier transfection process the Cytotoxic detection of Hela.Fig. 7 adopts in MTT colorimetric determination cationic liposomal gene of the present invention carrier cell transfection process the Cytotoxic detection of A549.Result shows, and along with the increase of sucrose ester dosage, the cytotoxicity of carrier system reduces gradually.Under best transfection efficiency condition, the toxicity of liposome to Hela cell and A549 cell is all little, and cell survival rate is between 80% ~ 100%.
(4) cellular uptake research
By A549 cell seeding in 6 porocyte culture plates, every hole adds cell culture fluid (containing dual anti-and serum) 1mL, and concentration is about 4.0 × 10 6individual/hole, hatches 24h, make transfection day cell density reach 70 ~ 80%.
Be 3:1 compound by liposome and siRNA with mass ratio, cumulative volume 200 μ L is added in every hole, cultivates 30h.Wash once with DPBS, every hole adds 0.3mL trypsin digestion cell, after 1-2min, adds 0.8mL culture medium.After centrifugal, cell 0.4mLDPBS diluted, uses 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 of liposomees all can enter cell, and cellular uptake rate is between 30 ~ 80%.Wherein, liposome CDO14S(CDO14 and S070 mass ratio are 1:1) cellular uptake amount reaches 76%.
Biological assessment experiment in embodiment 17 body
(1) foundation of mouse model
Take the logarithm trophophase Luc-A549 cell, wash once with DPBS, add 3mL trypsinization, adding 5mL has serum and dual anti-PRMI1640, blows and beats gently, transfers in 15mL centrifuge tube, centrifugal 5min under 1200rpm.Remove supernatant, be diluted in 1.5mLPBS, make concentration reach 2 × 10 7cells/mL.After falling nude mice sterilization, plant in nude mice forelimb oxter subcutaneous, every only about 200 μ L, tumor grows to 100-200mm 3time, can use.
(2) the reticent Luciferase gene of siRNA is delivered
12 tumor-bearing mices are divided into 4 groups at random, often organize 3, be respectively blank group, CDO14S liposome transfection group, positive controls (Lipofectamine2000 transfection group).By liposome/siRNA with mass ratio 3:1 compound, consumption is siRNA8 μ g/20g, liposome 24 μ g/20g.Complex is through tail vein injection in tumor-bearing mice, and before transfection and after transfection, 24h, 48h, 72h measure and record nude mice body weight, survey tumor size, and calculate tumor volume.The expression of luciferase in living imaging detection bodies, nude mice abdominal cavity injected fluorescein 3mg/20g, freely after movable 15min with halothane anesthesia, then enter living imaging system and take pictures.With the clean optical density in mouse tumor before and after transfection than the effect representing RNAi in body.
Cationic-liposome R 18oRRR delivers siRNA, and in mouse tumor cell, the result of reticent luciferase gene as shown in Figure 9,24h after injecting lipid body CDO14S, and mouse tumor volume becomes large gradually, but in mouse tumor cell, luciferase fluorescence intensity reduces by 50%.Illustrate that the embedded cationic-liposome CDO14S of sucrose ester can lower the overexpression of luciferase gene specifically, efficiently.
(3) in vivo cytotoxicity research
By liposome/siRNA with mass ratio 3:1 compound, through tail vein injection in tumor-bearing mice, every injection in two days once, inject 5 times altogether.Administration every day pre-test Mouse Weight, observes the change that each group of mice body weight every day increases.Simultaneously test mice serum biochemical indicator, investigates the sucrose ester embedded cationic-liposome toxicity to mouse liver and kidney.
The body weight change of mice as shown in Figure 10, before injection complex, the body weight of mice is 21-23g, after injection complex in 9 days Mouse Weight in the trend of rising overally, the average weight of the 9th day mice reaches 25-28g, show the adaptation of mice to the liposome CDO14S of doses, liposome can not affect the normal growth of mice.Table 1 is mice serum biochemical indicator after transfection, after transfection, the creatinine of mice, blood urea nitrogen, glutamic oxaloacetic transaminase, GOT and glutamate pyruvate transaminase changes of contents are little, all in range of normal value, illustrate that the embedded cationic-liposome of sucrose ester of the present invention does not produce toxicity to the liver of mice and kidney.
Table 1 is the embedded cationic-liposome CDO14S of sucrose fatty acid ester and gene composite to the toxicity of mouse liver and kidney.
Table 1
。

Claims (8)

1. the embedded cationic liposomal gene carrier system of sucrose fatty acid ester, it is characterized in that: described sucrose fatty acid ester embedded cationic liposomal gene carrier system is made up of the embedded cationic-liposome of sucrose fatty acid ester and genetic stew, and the embedded cationic-liposome of described sucrose fatty acid ester is made up of cation lipoid and sucrose fatty acid ester.
2. the embedded cationic liposomal gene carrier system of sucrose fatty acid ester according to claim 1, is characterized in that: described sucrose fatty acid 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 Palmic acid or Semen Myristicae acid or erucic acid;
The hydrophile-lipophile balance value of described sucrose fatty acid ester is: HLB<1 or 1≤HLB≤16.
3. the embedded cationic liposomal gene carrier system of sucrose fatty acid ester according to claim 1 and 2, is characterized in that: described cation lipoid is peptide type cation lipoid, and structural formula is:
；
Or Gemini type cationic lipoid, structural formula is:
；
Or quaternary ammonium salt cationic lipoid, structural formula is:
；
Wherein: x is selected from 1 ~ 6; Y is selected from 1 ~ 8; R is selected from C 8-20alkyl, this alkyl comprises 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.
4. the embedded cationic liposomal gene carrier system of sucrose fatty acid ester according to claim 3, is characterized in that: described cation lipoid and the mass ratio of sucrose fatty acid ester are 1 ︰ 8 ~ 8 ︰ 1; The mass ratio of the embedded cationic-liposome of described sucrose fatty acid ester and genetic stew is 0.5 ︰ 1 ~ 32 ︰ 1.
5. the embedded cationic liposomal gene carrier system of sucrose fatty acid ester according to claim 1 or 2 or 4, is characterized in that: described genetic stew is plasmid DNA or siRNA.
6. the embedded cationic liposomal gene carrier system of sucrose fatty acid ester according to claim 3, is characterized in that: described genetic stew is plasmid DNA or siRNA.
7. a preparation method for the embedded cationic liposomal gene carrier system of sucrose fatty acid ester according to claim 1, is characterized in that: step is as follows:
The preparation of the embedded cationic-liposome of sucrose fatty acid ester:
(1) cation lipoid and sucrose fatty acid ester to be dissolved in chloroform or methanol to obtain solution according to mass ratio 1:8 ~ 8:1 ratio;
(2) solution (1) step obtained blows out homogeneous film under a nitrogen, vacuum drying 2 ~ 12h;
(3) carry out aquation with ethanol or water or normal saline or phosphoric acid as buffer or with the mixed liquor of the quality such as any two kinds in ethanol, water, normal saline, phosphoric acid, hydration temperature is 10 ~ 80 DEG C; Hydration time is 1 ~ 10h, and ultrasonic vibration is to clarification, and obtain the embedded cationic-liposome of sucrose fatty acid ester, concentration is 0.5 ~ 3.0mg/mL; The embedded cationic-liposome of this sucrose fatty acid ester is be dispersed in the cationic-liposome of surface with positive charge that the size formed in aqueous phase is the stable homogeneous of 150 ~ 250nm;
The embedded cationic-liposome of sucrose fatty acid ester and genetic stew is utilized to prepare the embedded cationic liposomal gene carrier system of sucrose fatty acid ester:
By embedded for sucrose fatty acid ester cationic-liposome and plasmid DNA or siRNA, formed the nano-particle being dispersed in stable homogeneous in aqueous phase by electrostatic interaction, concrete steps are as follows:
(1) getting the embedded cationic-liposome of sucrose fatty acid ester is dispersed in cell culture fluid DMEM or RPMI1640, and mixing, makes concentration be 0.01 μ g/ μ L ~ 0.64 μ g/ μ L;
(2) be diluted in cell culture fluid DMEM or RPMI1640 by 0.5 ~ 1.0 μ g plasmid DNA or siRNA, mixing, makes plasmid concentration be 0.02 μ g/ μ L;
(3) according to the embedded cationic-liposome of sucrose fatty acid ester and genetic stew mass ratio 0.5:1 ~ 32:1 ratio, by (1) and the mixing of (2) two diluents, room temperature places 10 ~ 40min, can obtain the embedded cationic liposomal gene carrier system of sucrose fatty acid ester.
8. the application of the embedded cationic liposomal gene carrier system of sucrose fatty acid ester prepared of the preparation method of a sucrose fatty acid ester as claimed in claim 7 embedded cationic liposomal gene carrier system in cell transfecting and the application in vivo in transfection.
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