CN108524450A - Oxaliplatin and MDC1-AS based on magnetic temperature-sensitive cationic-liposome transmit the preparation and application of pharmaceutical carrier altogether - Google Patents
Oxaliplatin and MDC1-AS based on magnetic temperature-sensitive cationic-liposome transmit the preparation and application of pharmaceutical carrier altogether Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
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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
- A61K47/02—Inorganic compounds
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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
- A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
- A61K9/0009—Galenical forms characterised by the drug release technique; Application systems commanded by energy involving or responsive to electricity, magnetism or acoustic waves; Galenical aspects of sonophoresis, iontophoresis, electroporation or electroosmosis
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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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
This application discloses preparation and its application that a kind of oxaliplatin based on magnetic temperature-sensitive cationic-liposome and MDC1 AS transmit pharmaceutical carrier altogether, preparation methods：Magnetic temperature-sensitive cationic-liposome is prepared using film dispersion aquation method, the plasmid of oxaliplatin (OXA) and coding MDC1 antisense long-chains non-coding RNA (MDC1 AS) is fitted into this pharmaceutical carrier of transmission altogether, then measures vitro cytotoxicity and the internal antitumor activity of external OXA temperature-sensitives release activity, the ability of MDC1 AS regulation and control MDC1 and OXA and MDC1 AS respectively.As a result：This transmission system altogether has satisfactory targeted delivery efficiency, the ability of OXA temperature-sensitives releasability and MDC1 AS regulation and control MDC1.Moreover, compared with independent medication, the total transmission of OXA and MDC1 AS shows that more good inside and outside inhibits the activity of cervical cancer cell growth.Conclusion：The magnetic temperature-sensitive cationic liposome system that this novel OXA and MDC1 AS are transmitted altogether will have applications well foreground in Chemotherapy of Cervical Cancer and gene therapy synergy.
Description
Technical field
The present invention relates to a kind of liposomes, in particular to magnetic temperature-sensitive cationic-liposome (to have oxaliplatin and MDC1-
AS is total to transfer function) preparation and its targeted therapy cervical carcinoma application.
Background technology
Cervical carcinoma is a kind of most common female malignant, and in developing country, incidence and case fatality rate are in women
The 2nd is occupied in tumour, and incidence is increased with the speed of annual 2%-3%, seriously threatens the health of women.Operation, radiotherapy,
The conventional method that chemotherapy etc. is treated as gynecologic malignant tumor, wherein chemotherapy are to treat one of the Critical policies of cervical carcinoma.But
Traditional chemotherapy therapeutic effect in clinical application is poor, and side effect is big.For example：Oxaliplatin (OXA) is in kinds of tumors model system
System, is included in Human colorectal carcinoma model, all shows vitro cytotoxicity and the effect of internal antitumor activity of wide spectrum, but
There are neurotoxicity, cardiotoxicity, stomach and intestine abnormal response, massive haemorrhage, the serious adverse reactions such as allergic reaction.This be mainly because
It is mostly Formulations for systemic administration for classic chemotherapy, lacks targeting and controlled capability, effective drug concentration can not be reached in tumor by local, together
When relatively higher systemic blood levels can then cause many adverse reactions.
Chemotherapeutics will also experience the effect of drug resistance of tumor cell gene, so as to cause classic chemotherapy antitumous effect very
It is limited.Therefore, multi-agent kind co-administered system is come into being, because it can convey two distinct types of drug to same simultaneously
One tumour cell plays synergistic antitumor effect.Such as the chemotherapeutics or a kind ofization of two kinds of different role mechanism of conveying
It treats drug and combines the long-chain non-coding RNA (lncRNA) for being directed to certain proto-oncogene or tumor suppressor gene.Pass through combined gene therapy
Play the role of synergistic antitumor with chemotherapy.In addition, targeted delivery and drug controlled release can further promote antitumous effect
With reduction side effect.
Research shows that antisense lncRNAs is a kind of transcript from protein coding gene antisense strand.Several cancers are related
Gene, as ADAMTS9 and Myc has been proved to by their antisense lncRNAs regulation and control.It is ground according to Faghihi etc. is previous
Study carefully report：The regulatory mechanism of antisense lncRNAs, which is it, to exempt from the mRNA of contiguous gene formation RNA-RNA dimers protections mRNA
It is degraded by RNA enzyme, to increase the stability of mRNA and promote the expression of encoding gene.Xue in 2015 etc. has found MDC1-AS
It is lowered in carcinoma of urinary bladder with the expression of MDC1.After MDC1-AS is overexpressed, MDC1 expresses increase in transitional cell bladder carcinoma cell line.
They are it has also been found that MDC1-AS can significantly inhibit the deterioration of transitional cell bladder carcinoma cell line EJ and T24.In 2016, the researchs such as Yue were found
The expression of glioma MDC1-AS and MDC1 are significantly lowered compared with the control group, and the expression of the two is positively correlated.
MDC1-AS is overexpressed the apparent cell proliferation for inhibiting U87MG and U251 and cell cycle.These results and the MDC1 being previously reported
Low expression is consistent in tumor tissues, so, MDC1-AS is a kind of potential quality target spot for the treatment of of human cervical cancer.
Liposome (LP) is a kind of excellent drug or nucleic acid (that is, DNA and RNA) carrier, can be with according to its different characteristics
There are many types, wherein magnetic thermal sensitive liposome (magnetic thermosensitive liposomes, MTSLs) conduct
A kind of novel targeting drug delivery system, it is that magnetisable material such as Fe is added in thermal sensitive liposome2O3Or Fe3O4Again plus medicine
It is made.After vivo medicine-feeding, using magnetic nano-particle as heating element, fat is reached by external alternating magnetic field (AMF) heat production
Plastid phase transition temperature achievees the effect that Drug controlled release in tumor tissues targeting, multiple, pulse administration, to overcome
The generation of certain serious adverse reactions to improve the compliance and life quality of Tumor Patients after Chemotherapy there is good clinic to answer
With value.In addition, cationic-liposome be by positively charged cation lipid absorb negatively charged nucleic acid (such as DNA and
RNA) the liposome formed, this liposome is due to being a kind of good gene delivery load with low toxicity and immunogenicity
Body, therefore cationic-liposome can realize that the gene of cancer cell is controlled by the plasmid transfection to cancer cell for encoding LncRNAs
It treats.But in the prior art, the liposome for having not been reported while having magnetic temperature-sensitive and cationic overall characteristic, does not have yet
There are the medicine-carried system realized using the liposome and to drug or nucleic acid carry medicine function simultaneously, therefore, present inventor couple
This proposes to improve, and proposes the technical solution of the application.
Invention content
Technical problem to be solved of the embodiment of the present invention is, provides a kind of based on magnetic temperature-sensitive cationic-liposome
Oxaliplatin and MDC1-AS transmit the preparation method of pharmaceutical carrier and its inspection method of targeted therapy cervical carcinoma effect altogether.
As the first aspect of the invention purpose, the technical solution of the application provides a kind of magnetic temperature-sensitive cationic lipid
The preparation method of plastid.
As the second aspect of the invention purpose, the technical solution of the application provides a kind of oxaliplatin and MDC1-
AS transmits the preparation method of medicament carrier system altogether.
As the third aspect of the invention purpose, the present invention also provides a kind of based on the oxaliplatin and MDC1-
AS transmits the application of medicament carrier system targeted therapy cervical carcinoma altogether.
Realize the first aspect of the invention purpose, technical solution is according to dipalmitoylphosphatidylcholine：DC- courages are solid
Alcohol:Octadecyldimethyl ammonium bromide：Cholesterol=80:5:5:10 molar ratio weighing, then the total 20mg of phosphatide is taken, it is put into anti-
It answers in container, it is 2 that 10mL volume ratios, which are added,:1 chloroform-methanol dissolving, 37 DEG C are evaporated under reduced pressure one hour, make reaction container bottom
Form one layer of immobilized artificial membrane；Then the ferriferrous oxide nano grain and ammonium sulfate suspension hydrated films of 1mL is added, obtains magnetic temperature-sensitive
Cationic-liposome, the cation come from ammonium ion in ammonium sulfate.
Realize that the second aspect of the invention purpose, technical solution are that a kind of oxaliplatin and MDC1-AS transmit medicine altogether
The preparation method of object carrier system includes following steps：
(1) magnetic temperature-sensitive cationic-liposome is prepared by preparation method described in claim 1；
(2) it is then squeezed by polycarbonate filter and chemotherapeutics is encapsulated into liposome by ammonium sulphate gradient；Again
Non-encapsulated Fe is removed by centrifuging3O4The magnetic temperature-sensitive cationic-liposome for being mounted with oxaliplatin is formed, it then, will
PcDNA-LncRNA MDC1-AS plasmids and the magnetic temperature-sensitive cationic-liposome of chemotherapeutics is mounted in serum free medium
In be incubated at room temperature 30 minutes, transmit medicament carrier system altogether to prepare oxaliplatin and MDC1-AS.
The present invention also provides it is a kind of based on the oxaliplatin and MDC1-AS transmit altogether medicament carrier system targeting control
Treat the application of cervical carcinoma.
The magnetic temperature-sensitive cationic-liposome of the present invention is chemotherapy that is a kind of while having magnetic target tropism and temperature-sensitive controlled capability
The total transmission carrier of the plasmid of drug and coding LncRNAs can not only transmit chemotherapeutics and encode the matter of LncRNAs simultaneously
Grain, and realization tumor by local magnetic targeted orientation transport and temperature-sensitive under the action of directional magnetic field in vitro and alternating magnetic field can be distinguished
Trigger medicine controlled releasing.
This total liposome for carrying medicine and drug-loading system of transmitting of the present invention has satisfactory targeted delivery efficiency, OXA
The ability of temperature-sensitive releasability and MDC1-AS regulation and control MDC1.Moreover, compared with independent medication, the total transmission of OXA and MDC1-AS
Show that more good inside and outside inhibits the activity of cervical cancer cell growth, what this novel OXA and MDC1-AS was transmitted altogether
Magnetic temperature-sensitive cationic liposome system will have applications well foreground in Chemotherapy of Cervical Cancer and gene therapy synergy.
It is specifically shown in specification embodiment experimental data part.
Description of the drawings
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technology description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention, for those of ordinary skill in the art, without having to pay creative labor, according to
These attached drawings obtain other attached drawings and still fall within scope of the invention.
Fig. 1 oxaliplatins and MDC1-AS transmit the preparation flow figure of medicament carrier system, Tu1AZhong, MTCL-OXA-altogether
The preparation flow of lncRNA MDC1-AS:The structure of pcDNA-LncRNA MDC1-AS:Total serum IgE is extracted from SiHa cells, and
Synthesis cDNA is inverted as template.Then secondary segment is cloned into pcDNA 3.1 or pcDNA by then PCR amplification MDC1-AS
In 3.1-EGFP plasmids；Figure 1B is the preparation flow of MTCL-OXA-lncRNA MDC1-AS；
Used primer sequence table figure in Fig. 2 RT-PCR analyses；
The comparison of OXA temperature-sensitives release rate in Fig. 3 different liposomes, after being incubated in 37 DEG C and 42 DEG C of PBS respectively, different fat
OXA in plastid (TCL-OXA, MTCL-OXA, TCL-OXA-LncRNA MDC1-AS, MTCL-OXA-LncRNA MDC1-AS)
Temperature-sensitive discharges.In triplicate, value adds standard deviation to indicate with mean for each experiment.When temperature is increased to 37 DEG C, TCL-OXA and
For MTCL-OXA in PBS at 60 minutes, the release rate of OXA is 19% and 18% respectively.Meanwhile TCL-OXA-LncRNA
For MDC1-AS and MTCL-OXA-LncRNA MDC1-AS in PBS at 60 minutes, the release rate of OXA is 14% He respectively
16%.And when temperature is increased to 42 DEG C, in PBS at 60 minutes, the release rate of OXA is distinguished by TCL-OXA and MTCL-OXA
Increase to 42% and 44%.Meanwhile TCL-OXA-LncRNA MDC1-AS and MTCL-OXA-LncRNA MDC1-AS are in PBS
At 60 minutes, the release rate of OXA increases respectively to 39% and 40%；
Fig. 4 MTCL-LncRNA MDC1-AS are to the influence result figure of SiHa cell proliferations and apoptosis, Fig. 4 A.TCL-
After lncRNA MDC1-AS and MTCL-lncRNA MDC1-AS transfection SiHa cells, the SiHa cells under fluorescence microscope；Figure
After 4B.TCL-lncRNA MDC1-AS and MTCL-lncRNA MDC1-AS transfections, the increment of SiHa cells changes.Each experiment
In triplicate, the value measured adds standard deviation to indicate with mean.Compared with 24 hours groups of TCL-lncRNA MDC1-AS, * P<
0.05,*○P<0.01；Compared with 24 hours groups of MTCL-lncRNA MDC1-AS,#P<0.05,#△P<0.01.Fig. 4 C.TCL-
After lncRNA MDC1-AS and MTCL-lncRNA MDC1-AS transfections, the variation of SiHa Apoptosis.Each experiment repeats three
Secondary, the value measured adds standard deviation to indicate with mean.Compared with mock groups, * P<0.05,*#P<0.01；
The principle and as a result, Fig. 5 A of Fig. 5 Transwell experiments:The schematic diagram of Transwell Matrigels:Tumour cell
It is incubated at upper chamber, fetal calf serum is added in lower room, and then, tumour cell enters lower room full of nutrition from upper chamber.In makrolon
The upper chamber of film spreads one layer of matrigel and illustrates there is matrix metal if cell can enter lower room with analogue body extracellular matrix
Protease is secreted and degrades hypothallus, therefore by polycarbonate membrane, the cell quantity into lower room can reflect that tumour is thin
The invasive ability of born of the same parents, Fig. 5 B, Fig. 5 C:It is right after detection OXA, LncRNA MDC1-AS and the two are combined to be tested using Transwell
SiHa cell migrations (Fig. 5 B) and the ability for invading (Fig. 5 C).The value measured adds standard deviation to indicate (n=3) with mean.With compare
Group compares, * P<0.05,*●P<0.02,*●▲P<0.01,*●▲#P<0.005；
Schematic diagram and internal anti-tumor experiment result figure is administered in Fig. 6 cervical carcinoma nude mices, wherein Fig. 6 A. lotus cervical carcinoma nude mices
Brief introduction is administered:Lotus cervical carcinoma nude mice passed through tail vein injection saline, OXA, TCL-OXA, TCL- respectively every three days
LncRNA MDC1-AS, TCL-OXA-LncRNA MDC1-AS and MTCL-OXA-LncRNA MDC1-AS.After per injection,
Tumor surface applies the external magnetic field of 30 minutes 5000GS to realize the magnetic targeted of drug.Then, alternating magnetic field (H=12KA/m,
F=250kHz under) acting on, the temperature of tumor locus increases to 42 DEG C, to realize that the temperature-sensitive of drug discharges.Whole experiment process
In, above-mentioned treatment carries out three times altogether.The cartoon figure of the administration strategy of Fig. 6 B. lotus cervical carcinoma nude mices.Resist in Fig. 6 C, Fig. 6 D. bodies swollen
Tumor tests (Fig. 6 C. transplantable tumor sizes, Fig. 6 D. survivorship curves)；
The expression of Fig. 7 cervical carcinoma apoptosis-related genes, wherein Fig. 7 A, Fig. 7 B, Fig. 7 C:It is thin that RT-qPCR detects every group of SiHa
The expression of MDC1 in born of the same parents, BAX and Bcl-2；Fig. 7 D:Western Blot detect MDC1, BAX and Bcl-2 in every group of SiHa cell
Expression.In triplicate, the value measured adds standard deviation to indicate using mean for each experiment.Note：Ctrl represents Control, TCLO
TCL-OXA are represented, TCLM represents TCL-LncRNA MDC1-AS, and TCLOM represents TCL-OXA-LncRNA MDC1-AS, MTCLOM
Represent MTCL-OXA-LncRNA MDC1-AS.Compared with the control group, * P<0.05,**P<0.02,***P<0.01,****P<
0.005；
Specific implementation mode
To make the object, technical solutions and advantages of the present invention clearer, the present invention is made into one below in conjunction with attached drawing
Step ground detailed description.
The drug-loading system of this targeted therapy cervical carcinoma, by thermal sensitive liposome, nano-magnetic substance and cationic materials composition,
Thermal sensitive liposome selects temperature-sensitive phosphatide, liposome interior to be encapsulated with chemotherapeutics oxaliplatin and nano ferriferrous oxide particle,
Surface carries the nucleic acid containing gene therapy cervical carcinoma by the electrostatic adsorption of the cation lipid in lipid bilayer
Carrier.Temperature-sensitive phosphatide, nano ferriferrous oxide particle constitute magnetic temperature-sensitive cationic-liposome with cationic materials.
The oxaliplatin and MDC1-AS of the present invention transmits medicament carrier system also abbreviation MTCL-OXA-lncRNA altogether
MDC1-AS。
The MDC1 antisense long-chain non-coding RNAs of cervical cancer cell：MDC1-AS is a kind of potential quality target spot for the treatment of of human cervical cancer.
LncRNAs is a kind of transcript from protein coding gene antisense strand, the MDC1 antisense long-chains containing coding cervical cancer cell
Non-coding RNA：The plasmid of MDC1-AS is then the nucleic acid carrier as gene therapy cervical carcinoma, this drug-loading system, which uses, contains palace
The plasmid of the MDC1 antisense long-chain non-coding RNA transcript LncRNA MDC1-AS of neck cancer cell.
Chemotherapeutics entrained by this drug-loading system is not limited to the platinum class anti-cervical cancer medicine such as oxaliplatin；The nucleic acid, packet
Include RNA and DNA；The thermal sensitivity liposome, is not limited to the liposomes such as temperature-sensitive phosphatide；Nano-magnetic substance is not limited to nanometer four
Fe 3 O particle.The above variation is fallen in the scope of the present invention.
By the embodiment for preparing this drug-loading system hereafter, the implementation of the present invention, but this can be more fully understood
Invention is not limited to embodiments described just below.
1, the present embodiment brief introduction：
The present embodiment from thermal sensitivity, ground by magnetic responsiveness and three aspects of transfection abilities of plasmid for encoding LncRNAs
Study carefully, completes the structure of this novel supports；Then the plasmid of OXA and coding LncRNAs are packed into carrier, to realize that gene is controlled
Treat the synergy with chemotherapy., respectively from OXA temperature-sensitive release rates, MDC1-AS regulates and controls the ability of MDC1, OXA and MDC1-AS for we
Magnetic targeted transmission efficiency, vitro cytotoxicity effect and apoptosis induction experiment, tumor inhibition and real time fluorescent quantitative RT-
Its therapeutic effect to cervical carcinoma of PCR and Western Blot experimental checks.
2, materials and methods
SiHa cell strains be purchased from ATCC (Manassas, USA), cell culture containing 10% fetal calf serum FBS (Sigma,
In USA), 100U/mL penicillin, and the DMEM culture mediums of 50 μ g/mL streptomysins (Mediatech, USA) (Invitrogen,
USA), and it is incubated in containing 5%CO237 DEG C of incubators in.DMEM culture mediums are a kind of trainings containing various amino acid and glucose
Base is supported, is developed on the basis of MEM culture mediums.Various composition dosage is relatively increased with MEM, while being divided into as high glycoform
(being less than 4500mg/L) and low-sugar type (being less than 1000mg/L).High glycoform is conducive to cell and berths in a position growth, is suitable for
Very fast, more difficult tumour cell of attachment of growth etc..DMEM is the abbreviation of dulbecco's modified eagle medium, institute
Include mainly following with its feature:(1) amino acid content is 2 times of Yi Geer culture mediums, and contains nonessential amino acid, such as sweet
Propylhomoserin etc.；(2) vitamin content is 4 times of Yi Geer culture mediums；3) containing the important substance in glycolytic pathway -- pyruvic acid；
(4) contain micro iron ion.
The BALB/Ca nu/nu female mices (18-20g) of the athymia of six week old are purchased from Chinese Academy of Sciences Shanghai experimental animal
The heart.BALB/Ca nu/nu mouse are raised in the SPF grades environment (excluding other pathogens interference environment) of constant temperature, provide 12 hours：
The rodent of 12 hours illumination/dark cycles, standard is in kind, and free water.All zooperies are cured by Wenzhou
Section's university animal and Institutional Review Board are approved of and assessment (Wenzhou Medical University's animal policy and the welfare committee, document number:
WMU-2011-AP-0013)。
The reagent of selection is with consumptive material：Dipalmitoylphosphatidylcholine (DPPC) and DC-cholesterol are purchased from Avanti
Polar Lipids(USA).Cholesterol, octadecyldimethyl ammonium bromide (DOAB), triton x-100 and oxaliplatin (OXA)
It is purchased from Pharmacia Fine Chemicals purchased from Sigma-Aldrich (Saint Louis, USA) sephadex Gs -50
(Uppsala,Sweden).The four three body nanoparticles of oxidation that average diameter is 10 nanometers are purchased from Southwest China magnetic Applied Research Laboratory
(Chengdu, China).3.1 plasmids of pcDNA and pcDNA 3.1-EGFP plasmids are purchased from Invitrogen (Carlsbad, USA).
Cell Count Kit-8 are purchased from colleague's chemistry institute (Kyushu, Japan).AnnexinV–FITC/Propidium
Iodide apoptosis detection kits are purchased from Abcam (Cambridge, UK).2000 transfection reagents of Lipofectamine, Trizol
Reagent, DNA enzymatic I,III reverse transcriptases, PCR Master Mix, and SYBR Green PCR Master
Mix is purchased from Invitrogen (Carlsbad, USA).BCA protein quantification kits are purchased from Pierce (Rockford, USA).
Pvdf membrane is purchased from the laboratories Bio-Rad (Hercules, USA).Rabbit-anti people MDC1, Bcl-2, and Bax are purchased from Abcam
(Cambridge,UK).Goat-anti rabbit HRP IgG and Enhanced chemiluminescence (ECL) kit are purchased from Amersham Pharmacia
Biotech(Piscataway,USA).Other chemical reagent are the analytical reagents of business level.
The preparation of 2.1 MTCL-OXA-LncRNA MDC1-AS:
Total serum IgE is extracted from SiHa cells with Trizol first, is used by template of total serum IgEIII is reversed
Record enzymatic synthesis cDNA, then using GAPDH as internal reference, PCR amplification MDC1-AS full length fragments, wherein primer sequence reference literature
It designs (being shown in Table 1).Finally pcr amplification product MDC1-AS full length fragments are cloned into 3.1 plasmids of pcDNA or pcDNA 3.1-
EGFP plasmids are to build MDC1-AS expression plasmids (see Figure 1A).
Table 1:Each gene primer sequence in RT-PCR analyses
According to dipalmitoylphosphatidylcholine：DC-cholesterol:Octadecyldimethyl ammonium bromide：Cholesterol=80:5:5:
The 10 total 20mg of molar ratio weighing phosphatide, is put into round-bottomed flask, and 10mL chloroform-methanols (2 are added:1, volume ratio) dissolving；37
DEG C be evaporated under reduced pressure one hour, make round-bottomed flask bottom formed one layer of immobilized artificial membrane；Then the Fe of 1mL is added3O4With ammonium sulfate suspension water
Change film, is then squeezed by polycarbonate filter and OXA is encapsulated into liposome by ammonium sulphate gradient；Pass through centrifugation again
(1,000 × g, 15min) removes non-encapsulated ferriferrous oxide nano particle shape into magnetic temperature-sensitive cationic-liposome-Ao Shali
Platinum.Finally, pcDNA-LncRNA MDC1-AS plasmids and magnetic thermal sensitive liposome-oxaliplatin room temperature in serum free medium
It is lower to be incubated 30 minutes, medicament carrier system, abbreviation MTCL-OXA-LncRNA are transmitted altogether to prepare oxaliplatin and MDC1-AS
MDC1-AS, being specifically translated as Chinese is：The C1 antisenses of magnetic temperature-sensitive cationic-liposome-oxaliplatin-MD cervical cancer cells are long
Chain non-coding RNA transcript MTCL-OXA-LncRNA MDC1-AS, set in 4 DEG C of refrigerators and preserve, be specifically shown in Figure 1B.
2.2 forms and biophysical characteristics:
It after taking appropriate MTCL-OXA-LncRNAMDC1-AS to be diluted with distilled water, drops on special spray film copper mesh, stands
1min, with 3% phosphotungstic acid negative staining, standing 20s makes particle in copper deposited thereon, and copper mesh edge surplus liquid is sucked with filter paper, uses
Transmission electron microscope observing particle size and form (note：In above-mentioned steps, copper mesh then observable TCL-OXA and TCL-are not had to
The particle size and form of LncRNA MDC1-AS).
Take 50 μ l of Liposomal suspensions；2ml distilled waters are added, fully dilute；Liposomal suspensions after dilution are poured into colorimetric
In ware, it is placed in laser diffraction analyzer；25 DEG C of grain sizes and polydispersity using dynamic light scattering principle detection liposome；Profit
Surface potential is detected with electrophoretic mobility.
Quantitative and encapsulation rate the measurement ginseng of OXA in TSCL-OXA, TSCL-AEG-1siRNA and TSMCL-AEG-1siRNA
Examine document (Qingsheng Xie, Jinxiao Liang, Qunxian Rao, Xiaofei Xie, Ruixin Li, Yunyun
Liu,Hui Zhou,Jingjing Han,Tingting Yao,Zhongqiu Lin.Aldehyde Dehydrogenase
1Expression Predicts Chemoresistance and Poor Clinical Outcomes in Patients
with Locally Advanced Cervical Cancer Treated with Neoadjuvant Chemotherapy
Prior to Radical Hysterectomy.Annals of Surgical Oncology.2016,23(1):163-70)
It is described.
The measurement of the temperature-sensitive release rate of 2.3 OXA liposomes:
OXA Liposomal suspensions are analyzed respectively in 37 DEG C and 42 DEG C of drug release behavior using dialysis：It will contain respectively
The dialysis tubing (14,000MWCO) of 1mL oxaliplatin liposome suspensions is transferred in the beaker of the 37 DEG C and 42 DEG C PBS containing 50mL,
And continue to stir with the rotating speed of 100rpm.Then remove 1mL's from beaker respectively and at 0,5,10,15,30,60,90 minute
Then liquid supplements the PBS of equivalent.The total volume high pressure liquid chromatographic analysis of OXA in liposome, experiment is in triplicate.
2.4 cell culture and transfection efficiency detection：
SiHa cell culture passes on postposition in the DMEM culture mediums containing 10% fetal calf serum, through 0.25% trypsin digestion
It is cultivated in 37 DEG C of incubators containing 5%CO2, per 2-3d, passage is primary.The day before transfection, by SiHa cells with 2x105A cell/
The density in hole is inoculated in 12 porocyte culture plates, and growth is overnight to 90% fusion；Before transfection, culture medium is exhausted, cell is used
The sterile PBS of preheating is washed twice；By control group (that is, the mixing of pcDNA3.1-EGFP empty plasmids and Lipofectamine 2000
Object), TCL-LncRNA MDC1-AS (see 2.1) groups and MTCL-LncRNA MDC1-AS (see 2.1) group are separately added into 12 orifice plates
In, the external magnetic field 30 minutes of 5000GS is then added in 12 hole board bottoms.After transfection 6 hours, culture solution is replaced with containing 10%FBS
DMEM complete mediums, be put into cell incubator continue culture 48 hours.It is detected in SiHa cells by fluorescence microscope
The expression of EGFP assesses transfection efficiency.
2.5 CCK-8 detect the cell Proliferation of MTCL-LncRNA MDC1-AS：
Logarithmic growth phase cell is inoculated in 96 orifice plates with 5000/hole (100 μ l), and 3 groups of cell point is tested：It is right
According to group (that is, mixture of pcDNA3.1 empty plasmids and Lipofectamine 2000), TCL-LncRNA MDC1-AS combinations
MTCL-OXA-LncRNA MDC1-AS groups, every group of 5 multiple holes.After corresponding reagent is added to each group, add at 96 well culture plate bottoms
The external magnetic field of 5000GS is simultaneously warming up to 42 DEG C of holding 30min.After transfection 6 hours, 100 μ l are added per hole and contain 10% fetal calf serum
Culture medium continues to cultivate.The training of the reagent containing 10%CCK-8 is changed to after 24 hours, 48 hours and 72 hours respectively at pharmaceutical intervention
Foster base continues culture 2 hours.Blank control wells (10 μ l CCK-8 reagents are added in not celliferous culture medium) return to zero, with enzyme mark
Instrument measures absorbance (A) value (reference wavelength 600nm) at 450nm wavelength, and all experiments are in triplicate.Cell inhibitory rate=
(1- experimental groups A values/blank control group A values) x100%.
The Apoptosis of 2.6 MTCL-LncRNA MDC1-AS detects:
Experiment packet is same as above, and is inoculated in after cell dissociation in 6 well culture plates, after corresponding reagent is added in each group.It is trained in 12 holes
Foster board bottom adds the external magnetic field of 5000GS and is warming up to 42 DEG C of holding 30min.Then cell culture digests after 48 hours, centrifuges respectively
Collect above-mentioned 3 groups of cell 2X105A, PBs rinsings are resuspended in 500ul combination buffers afterwards twice.5 μ l AnnexinV- are added
FITC and 5 μ l propidium iodides (PI) dye liquors, room temperature are protected from light 30min, and machine measures each group apoptosis rate on flow cytometer.
2.7 cells Transwell migrate and Matrigel:
The SiHa cells of exponential phase are with 2x105A cells/well is inoculated in 12 orifice plates, 6 groups of experiment point：Control group is (no
Add any reagent), OXA, TCL-OXA, TCL-LncRNA MDC1-AS, TCL-OXA-LncRNA MDC1-AS and MTCL-OXA-
LncRNA MDC1-AS.The dosage that the dosage of OXA is 20 μ g/mL, pcDNA-LncRNA MDC1-AS is 1.5 holes μ g/.Each group adds
After entering corresponding reagent, adds 5000GS external magnetic fields in 12 hole board bottoms and be warming up to 42 DEG C 30 minutes.After being incubated 48 hours, each group cell
It is resuspended with the culture medium containing 1% serum.Particular content is as follows：The upper chamber of each cells Transwell is added 5 × 104Cell, under
The culture solution that 600 μ l contain 10% fetal calf serum is added in room, and when addition avoids the generation of bubble.It is placed in 5%CO2,37 DEG C, saturation is wet
After continuing culture for 24 hours under degree environment, culture solution in upper chamber is abandoned, PBS is rinsed 2-3 times, and being placed in 4% paraformaldehyde under room temperature fixes
15min.Upper indoor liquid is sucked with cotton swab, 1% crystal violet dye liquor dyes 15min at room temperature, and PBS is rinsed 2-3 times, under microscope
It takes 5 visuals field to take pictures at random per hole, count cell number and is averaged.Every group of cell is all provided with 3 multiple holes, and experiment is repeated 3 times.
The DMEM culture mediums of the Matrigel glue of precooling and serum-free are pressed 1:8 dilutions are laid on the cells Transwell or more
Between room, uniformly it is coated in the cells Transwell bottom film per 50 μ l of hole, sets incubator 30min and be allowed to plastic.Remaining step
It is identical (Fig. 5 A) as migration experiment.
The foundation of 2.8 animal models and tumor inhibition：
Balb/c nude mices are raised in the dedicated laminar flow animal room of Wenzhou Medical University's Experimental Animal Center；Take logarithmic growth
Single cell suspension is made in the SiHa cells of phase, and adjustment cell concentration is 1X107/ml；By 200 μ l pallium cell injections to nude mice
Help portion subcutaneous；Select the nude mice that growth of transplanted human is good, diameter of tumor is about 0.8cm or so as this experimental animal mould after 2 weeks
Type.
36 tumor-bearing mices are randomly divided into 6 groups (n=6)：Through tail vein difference injecting normal saline (control), OXA,
TCL-OXA, TCL-LncRNA MDC1-AS, TCL-OXA-LncRNA MDC1-AS and MTCL-OXA-LncRNA MDC1-AS；
The dosage of OXA is 2.5mg/kg weight, and pcDNA-LncRNA MDC1-AS are every 8 μ g of mouse.
It is primary (Fig. 6 A, 6B) every injection in 3 days, apply 30 minutes 5000GS at superficial tumor position after per injection
Directional magnetic field to realize the magnetic targeted of drug, then in alternating magnetic field (Alternating Magnetic Field H=
12KA/m, f=250kHz) under the action of make tumor focus local heating to 42 DEG C to realize drug temperature-sensitive release, altogether into
3 treatments of row, measure transplantable tumor size 1 time within every 2 days after treatment (the tumour maximum diameter a and minor axis b vertical with maximum diameter is measured,
Gross tumor volume V=ab2/ 2) it, puts to death animal within the 20th day after treatment, subcutaneously removes each animal-transplanted tumor, weigh tumor quality, then
Liquid nitrogen storage.Tumor control rate is calculated by document [5].
2.9 RT-QPCR detect the expression of cervical carcinoma apoptosis-related genes:
It uses Trizol reagents to extract tumor tissues total serum IgE first, then usesIII Reverse
Transcriptase reverse transcriptions go out cDNA, and real-time fluorescence quantitative PCR reaction uses SYBR Green PCR Master Mix roots
It is carried out according to specification.Each gene primer sequence is shown in Table 1.50 μ L of reaction system:DNA profiling (reverse transcription cDNA), primer, ddH2O,
25μL SYBR Green PCR Master Mix.Reaction condition is as follows:MDC1：94 DEG C of pre-degenerations 5 minutes, 35 cycles, 95
DEG C denaturation 30 seconds, 53 DEG C anneal 30 seconds, 68 DEG C extend 10 minutes, 4 DEG C preservation；Bcl-2：94 DEG C of pre-degeneration 5min, 35 cycles,
95 DEG C are denaturalized 30 seconds, and 58 DEG C are annealed 30 seconds, and 68 DEG C extend 10 minutes, 4 DEG C of preservations；BAX：94 DEG C of pre-degeneration 5min, 35 cycles,
95 DEG C are denaturalized 30 seconds, 60 DEG C of return of goods moneys 60 seconds (BAX), and 68 DEG C extend 10 minutes, 4 DEG C of preservations；GAPDH：94 DEG C of pre-degeneration 5min,
35 cycles, 95 DEG C are denaturalized 30 seconds, and 58 DEG C are annealed 30 seconds, and 68 DEG C extend 10 minutes, 4 DEG C of preservations.Every group is repeated 3 times, relatively more each
MRNA is with respect to internal reference changes of contents (target gene gray scale/internal reference gray scale) for group.
The expression of 2.10 Western blot detection cervical carcinoma apoptosis-related genes:
Tumor tissues albumen is extracted, BCA methods survey albumen concentration.Sample-loading buffer, SDS-PAGE electrophoresis, per porin is added
Content is about 50 μ g.By protein delivery to pvdf membrane, Ponceaux dyeing, the TBST containing 5% skimmed milk power is stayed overnight in 4 DEG C of closings.
It is added on pvdf membrane and is incubated 2h by rabbit-anti people MDC1, Bcl-2, and the BAX gene antibodies of respective dilution proportion, PBS washes film；
1: 1000 diluted goat-anti rabbit secondary antibody, 37 DEG C of incubation 2h, Enhanced chemiluminescence (ECL) colour developing is added.Observe each band depth
Change and analyzed, β-actin make internal reference.Band gray value is measured, with β-actin ratios as albumen relative expression
Amount.
2.11 statistical method：
Data acquired is all made of SPSS11.5 editions software packages and carries out statistical procedures, and more comparison among groups are examined using homogeneity of variance
Test with one-way analysis of variance (One Way ANOVA), with P ＜ 0.05 be difference it is statistically significant.
3 results:
The preparation of 3.1 MTCL-OXA-LncRNA MDC1-AS and feature:
The MDC1-AS full length fragments of PCR amplification are cloned into 3.1 carriers of pcDNA, to build pcDNA-LncRNA
MDC1-AS expression vectors.Amplified fragments confirm that its nucleotide sequence is completely correct through sequencing, this discovery shows pcDNA-
LncRNA MDC1-AS expression vectors have built success.
Transmission electron microscope shows MTCL substantially at smooth spherical structure, due to enclosing Fe in liposome membrane3O4Particle and
Dyeing is deeper.As shown in table 2, the grain size of TCL is 86.5 ± 9.3nm.Due to enclosing OXA, the grain size of TCL-OXA increases to
138.3±8.1nm(P<0.05), simultaneously, because having adsorbed pcDNA, TCL-LncRNA MDC1-AS and TCL-OXA-LncRNA
The grain size of MDC1-AS also dramatically increases (P<0.05).For magnetic liposome, MTCL, MTCL-OXA, MTCL-LncRNA
The grain size of MDC1-AS and MTCL-OXA-LncRNA MDC1-AS is ± 13.4 Hes of 140.6 ± 15.9,178.3 ± 16.1,243.0
350.5 ± 21.7nm, it is aobvious compared with TCL, TCL-OXA, TCL-LncRNA MDC1-AS and TCL-OXA-LncRNA MDC1-AS respectively
It writes and increases (P<0.05).The encapsulation rate of TCL-OXA and MTCL-OXA are 88.7% ± 7% and 78.2% ± 4% (n=3) respectively,
The encapsulation rate of TCL-OXA-LncRNA MDC1-AS and MTCL-OXA-LncRNA MDC1-AS is 83.4% ± 3% He respectively
70.1% ± 6%, prompt the absorption of LncRNA MDC1-AS plasmids not cause the leakage of OXA in liposome.
The grain size of 2. liposome of table, polydispersity and surface potential (n=3)
Note:*P<0.05, TCL-LncRNA MDC1-AS is to TCL；TCL-OXA-LncRNA MDC1-AS are to TCL-OXA；
MTCL-LncRNA MDC1-AS are to TCL；MTCL-OXA-LncRNA MDC1-AS are to MTCL-OXA.#P<0.05, MTCL to TCL；
MTCL-OXA is to TCL-OXA；MTCL-LncRNA MDC1-AS are to TCL-LncRNA MDC1-AS；MTCL-OXA-LncRNA
MDC1-AS is to TCL-OXA-LncRNA MDC1-AS.
The measurement of 3.2 OXA temperature-sensitive release rates：
As shown in figure 3, at 37 DEG C, 60 minutes, TCL-OXA and the MTCL-OXA release rate of OXA in PBS are respectively
19% and 18%, and the release of TCL-OXA-LncRNA MDC1-AS and MTCL-OXA-LncRNA MDC1-AS OXA in PBS
Rate is 14% and 16% respectively.The above results prompt TCL-OXA, MTCL-OXA, TCL-OXA-LncRNA MDC1-AS and MTCL-
OXA-LncRNA MDC1-AS keep stablizing at 37 DEG C, and the absorption of Plasmid DNA does not have significant impact (P for its stability>
0.05).When temperature rise is to 42 DEG C, in PBS at 60 minutes, the release rate of OXA increases separately by TCL-OXA and MTCL-OXA
To 42% and 44%.And TCL-OXA-LncRNA MDC1-AS and MTCL-OXA-LncRNA MDC1-AS are 60 minutes in PBS
When, the release rate of OXA is increased separately to 39% and 40%.TCL-OXA, MTCL-OXA, TCL-OXA-LncRNA MDC1-AS and
MTCL-OXA-LncRNA MDC1-AS are when 42 DEG C of OXA release rates are all remarkably higher than 37 DEG C.The above results are shown, are based on TCL
The liposome of prescription has good OXA temperature-sensitives release efficiency, can be used for carrying out the OXA temperature-sensitive controls of external alternating magnetic field triggering
It releases.
3.3 MTCL-LncRNA MDC1-AS rise in value to cervical cancer cell and the influence of apoptosis:
Transfection of this research respectively by control group, TCL-LncRNA MDC1-AS and MTCL-LncRNA MDC1-AS is real
Test the transfection efficiency for having rated MDC1-AS in cervical cancer cell.As shown in Figure 4A, pass through directional magnetic field outside MTCL combinations
Effect can significantly improve gene transfer efficiency (P<0.05).
The A values of each time point SiHa cell proliferations of each group, which are shown in Table 3, LncRNA MDC1-AS, obviously SiHa cell Proliferations
Inhibiting effect.Transfection for 24 hours, 48h and 72h when, TCL-LncRNA MDC1-AS inhibiting rates are respectively 7.7% ± 3.3%, 22.3%
± 2.2% and 30.0% ± 7.6%, MTCL-LncRNA MDC1-AS inhibiting rates are respectively 12.4% ± 2.9%, 31.5% ±
5.2% and 41.0% ± 6.1%.The inhibiting rate of MTCL-LncRNA MDC1-AS is apparently higher than TCL-LncRNA MDC1-AS (P<
And Mock (P 0.05)<0.01) inhibiting rate, 72 hours inhibiting effect are the most apparent (see Fig. 4 B).
Table 3.LncRNA MDC1-AS to SiHa cell proliferations influence (CCK-8A values,)。
Note:*P<0.05, compared with mock groups；#P<0.05, compared with TCL-lncRNA MDC1-AS groups.
Flow cytomery the results show that SiHa cells in Mock groups, TCL-OXA-LncRNA MDC1-AS groups and
The apoptosis rate of MTCL-OXA-LncRNA MDC1-AS groups is 2.80% ± 2.75% respectively, 15.79% ± 2.60% He
22.15% ± 3.56%.The apoptosis rate of MTCL-LncRNA MDC1-AS is apparently higher than TCL-LncRNA MDC1-AS (P<0.05)
With Mock (P<0.01) apoptosis rate (see Fig. 4 C).
3.4 the cells Transwell migrate and Matrigel:
Just as shown in Figure 5 B, it is 332 ± 59 that the cell migration number of control group, which is the cell migration number of 416 ± 52, OXA groups,
(compared with the control group, P<0.05), the cell migration number of TCL-OXA groups be 248 ± 27 (compared with OXA groups, P<0.05),TCL–
The cell migration number of LncRNA MDC1-AS groups be 260 ± 55 (compared with OXA groups, P<0.05),TCL–OXA–LncRNA
The cell migration number of MDC1-AS groups be 175 ± 36 (compared with TCL-OXA groups and TCL-LncRNA MDC1-AS groups, P<0.05),
The cell migration number of MTCL-OXA-LncRNA MDC1-AS groups is 110 ± 68 (with TCL-OXA-LncRNA MDC1-AS group ratios
Compared with P<0.05).
Equally, just as shown in Figure 5 C, it is 215 that the cell invasion number of control group, which is the cell invasion number of 280 ± 45, OXA groups,
± 31 (with control group ratio, P<0.05), the cell invasion number of TCL-OXA groups be 155 ± 51 (compared with OXA groups, P<0.05),
The cell invasion number of TCL-LncRNA MDC1-AS groups be 169 ± 57 (compared with OXA groups, P<0.05),TCL–OXA–LncRNA
The cell invasion number of MDC1-AS groups be 108 ± 20 (compared with TCL-OXA combinations and TCL-LncRNA MDC1-AS groups, P<
0.05), the cell invasion numbers of MTCL-OXA-LncRNA MDC1-AS groups is 70 ± 31 (with TCL-OXA-LncRNA MDC1-AS
Group compares, P<0.05).
The above results prompt MTCL-OXA-LncRNA MDC1-AS obviously inhibit the energy of migration and the invasion of SiHa cells
Power.
3.5 tumor inhibition：
By Nude Mouse Model, we examine control group, OXA groups, TCL-OXA groups, TCL-LncRNA MDC1-
AS groups, the internal antitumor activity of TCL-OXA-LncRNA MDC1-AS groups and MTCL-OXA-LncRNA MDC1-AS groups.Such as figure
Shown in 6C, the 20th day after pharmaceutical intervention, control group, OXA groups, TCL-OXA groups, TCL-LncRNA MDC1-AS groups, TCL-OXA-
LncRNA MDC1-AS groups and MTCL-OXA-LncRNA MDC1-AS groups be 2.26 ± 0.27,1.69 ± 0.16,1.24 ±
0.23,1.03 ± 0.17,0.839 ± 0.21 and 0.662 ± 0.3cm3。
In addition, as shown in Figure 6 D, by combination therapy, the median survival interval of mice with tumor is also obviously prolonged；The above results carry
Show that total transmission OXA and LncRNA MDC1-AS can generate the antitumor action of collaboration, the internal tumor suppression effect that combination therapy generates
Fruit, which is significantly stronger than, is used alone OXA or LncRNA MDC1-AS, and magnetic targeted can enhance antitumous effect.
Expression of 3.6 apoptosis-related genes in cervical carcinoma:
RT-qPCR results are shown：In each group, in MTCL-OXA-LncRNA MDC1-AS groups the MDC1 of tumor tissues and
BAX expression highest (that is, compared with the control group, P<0.01；Compared with OXA groups, P<0.01；Compared with TCL-OXA groups, P
<0.01；Compared with TCL-LncRNA MDC1-AS groups, P<0.01；Compared with TCL-OXA-LncRNA MDC1-AS groups, P<
0.05) (see Fig. 7 A and 7B).On the contrary, in each group, the Bcl-2 of tumor tissues in MTCL-OXA-LncRNA MDC1-AS groups
Expression it is minimum (that is, compared with the control group, P<0.01；Compared with OXA groups, P<0.01；Compared with TCL-OXA groups, P<
0.01；Compared with TCL-LncRNA MDC1-AS groups, P<0.01；Compared with TCL-OXA-LncRNA MDC1-AS groups, P<0.05)
(see Fig. 7 C).
Equally, Western blot results are shown：In each group, tumor group in MTCL-OXA-LncRNA MDC1-AS groups
The MDC1 and BAX that knit expression highest (that is, compared with the control group, P<0.01；Compared with OXA groups, P<0.01；With TCL-
OXA groups are compared, P<0.01；Compared with TCL-LncRNA MDC1-AS groups, P<0.01；With TCL-OXA-LncRNA MDC1-AS groups
It compares, P<0.05) (see Fig. 7 D).On the contrary, in each group, the Bcl-2 of tumor tissues in MTCL-OXA-LncRNA MDC1-AS groups
Expression it is minimum (that is, compared with the control group, P<0.01；Compared with OXA groups, P<0.01；Compared with TCL-OXA groups, P<
0.01；Compared with TCL-LncRNA MDC1-AS groups, P<0.01；Compared with TCL-OXA-LncRNA MDC1-AS groups, P<0.05)
(see Fig. 7 D).
4. conclusion：
A kind of completely new drug-loading system thermosensitive magnetism cationic-liposome (magnetic thermosensitive of the application
Cationic liposome, MTCL) in vitro under the action of directional magnetic field, OXA and LncRNA MDC1-AS carriers can be by target
To tumor locus, and in vitro under the action of alternating magnetic field, OXA can also be discharged (Fig. 3) in a manner of temperature-sensitive.Temperature-sensitive discharges
Pharmacological dependence is mainly made of dipalmitoylphosphatidylcholine (DPPC) in thermal sensitive liposome, this liposome, and DPPC can be sent out
Raw gel discharges (leakage) soluble small molecular drug at 41 DEG C to the phase transformation of mesomorphic state.By different DPPC and lipid
The liposome of composition has different phase transition temperature (Tm) and thermal sensitivity.Dsc analysis shows that our Tm of above-mentioned transmission system are
40.8 DEG C, and OXA releases are analysis shows that it at 37 DEG C is stable, and OXA significantly discharges when temperature is raised to 42 DEG C.This
Outside, LncRNA MDC1-AS are packed into temperature-sensitive release no significant impact of the temperature-sensitive cationic-liposome to OXA.So we
Liposome the satisfactory thermal sensitivity of configuration shows and also can be released for temperature-sensitive control the local heating in the state of
Put drug.However, temperature-sensitive release drug is derived from the infiltrative increase of liposome, rather than the rupture of liposome, and liposome
Although the middle composition that cholesterol is added can increase stability of the liposome in serum, by broadening phase transformation peak to liposome
Thermal sensitivity but has negative effect.So even if we obtain satisfactory thermal sensitivity, but drug release rate still compared with
It is low.
Magnetic target medicine (MDT) can promote drug in tumour under the guiding of directional magnetic field in vitro as a kind of pharmaceutical carrier
A large amount of accumulations of target area.Meanwhile gene transfer efficiency can also transfect to obtain with magnetic cation liposome by liposome magnetic
Improve.We devise the biography that MTCL-OXA-LncRNA MDC1-AS transfect synergy as a kind of MDT and magnetic in the application
Delivery system, to promote the transmission efficiency of OXA and LncRNA MDC1-AS.The fluorescence imaging experiments result prompt of cell transfecting passes
The increase for passing efficiency, which is that directional magnetic field guiding in vitro is lower, realizes (Fig. 4 A).The detection display of CCK-8 cell Proliferations：With Mock groups
It closes and TCL-LncRNA MDC1-AS groups compares, MTCL-LncRNA MDC1-AS can obviously inhibit SiHa cell Proliferations (figure
4B).Flow cytomery the result shows that, with Mock combination and TCL-LncRNA MDC1-AS groups compared with, MTCL-LncRNA
MDC1-AS can be obviously promoted SiHa Apoptosis (Fig. 4 C).The cells Transwell migrate and the prompt of Matrigel result：With
Other groups are compared, and MTCL-OXA-LncRNA MDC1-AS can significantly increase OXA and inhibit SiHa cell migrations and invasion activity
(Fig. 4 B, C).Finally, nude mice cervical carcinoma transplantable tumor animal experiment in vivo is shown under the guiding in magnetic field, OXA and LncRNA
The total transmission of MDC1-AS carriers presents the stronger effect (Fig. 6 C, 6D) for inhibiting tumour growth.RT-qPCR and Western
The expression of the experiment show of the Blot Bcl-2 of MTCL-OXA-LncRNA MDC1-AS is significantly lower than other groups
Expression, on the contrary, the expression of the BAX and MDC1 of MTCL-OXA-LncRNA MDC1-AS is apparently higher than other groups of table
Up to horizontal (Fig. 6).Generally speaking, these data confirm thats promote drug delivery efficiency and antitumor activity under the guiding in magnetic field
It can be achieved.
In conclusion novel magnetic temperature-sensitive cation drug-loading system transmits OXA and LncRNA MDC1-AS targeted therapies altogether
The mechanism of cervical carcinoma is following (see Fig. 8)：Thermosensitive magnetism cation drug-loading system first is structurally characterized in that in lipid bilayer
It is encapsulated with nano level ferroso-ferric oxide and chemotherapeutics, the cation lipid composition in lipid bilayer is connected with pcDNA-
LncRNAMDC1-AS plasmids；This thermosensitive magnetism cation drug-loading system has magnetic targeted effect, that is to say, that works as thermosensitive magnetism
Cationic-liposome (MTCL) can be gathered largely at tumor focus under the magnetic traction of some strength externally-applied magnetic field, be reduced
To the toxic side effect of healthy cell, to the active targeting of experimental drug；And thermosensitive magnetism cation drug-loading system also has
Temperature-sensitive release effects：For temperature by the effect of external alternating magnetic field from when being increased to 42 DEG C for 37 DEG C, triggering is hot at tumor focus
Drug is largely discharged in the quick magnetic cation liposome short time, to greatly increase the concentration of affected area drug, passes through collaboration
Effect is obviously improved therapeutic effect.In SiHa cells, the lncRNA MDC1-AS in MTCL can form RNA-RNA bis- with MDC1
Aggressiveness protects MDC1 from the degradation of RNA enzyme, to increase the stability of MDC1 and promote its expression.In addition, in MTCL
OXA can inhibit the reparation and transcription of DNA by forming platinum-DNA adduct (Pt-GG and Pt-AG) in chain with interchain,
To make BAX expression quantity increase and the reduction of Bcl-2 expression quantity, the increased activity of caspase-3 is then caused to lead to Siha cells
Apoptosis finally causes Siha cell deaths, to achieve the effect that synergistic treatment.
In short, we researches show that this novel drug and gene be total to transmission system (MTCLs) have it is good fixed
The joint of the temperature-sensitive controlled capability of magnetic target tropism, alternating magnetic field triggering under to magnetic fields and the inside and outside antitumor action of collaboration
Feature.So this temperature-sensitive cation magnetoliposomes are total to transmission system in the chemotherapy of cervical carcinoma and the joint of gene therapy
There is potential application in treatment.
The above disclosure is only the preferred embodiments of the present invention, cannot limit the right model of the present invention with this certainly
It encloses, therefore equivalent changes made in accordance with the claims of the present invention, is still within the scope of the present invention.
Claims (5)
1. a kind of preparation method of magnetism temperature-sensitive cationic-liposome, it is characterised in that：According to dipalmitoylphosphatidylcholine：
DC-cholesterol:Octadecyldimethyl ammonium bromide：Cholesterol=80:5:5:10 molar ratio weighing, then take phosphatide and be total to
20mg is put into reaction vessel, and it is 2 that 10mL volume ratios, which are added,:1 chloroform-methanol dissolving, 37 DEG C are evaporated under reduced pressure one hour, make
Reaction container bottom forms one layer of immobilized artificial membrane；Then the ferriferrous oxide nano grain and ammonium sulfate suspension hydrated films of 1mL is added,
Magnetic temperature-sensitive cationic-liposome is obtained, which comes from ammonium ion in ammonium sulfate.
2. the magnetic temperature-sensitive cationic-liposome prepared by a kind of preparation method as described in claim 1.
3. a kind of oxaliplatin and MDC1-AS transmit the preparation method of medicament carrier system altogether, it is characterised in that include following
Step：
(1) magnetic temperature-sensitive cationic-liposome is prepared by preparation method described in claim 1；
(2) it is then squeezed by polycarbonate filter and chemotherapeutics is encapsulated into liposome by ammonium sulphate gradient；Pass through again
Centrifugation removes non-encapsulated Fe3O4The magnetic temperature-sensitive cationic-liposome for being mounted with oxaliplatin is formed, then, by pcDNA-
LncRNA MDC1-AS plasmids and the magnetic temperature-sensitive cationic-liposome room temperature in serum free medium for being mounted with chemotherapeutics
It is lower to be incubated 30 minutes, medicament carrier system is transmitted altogether to prepare oxaliplatin and MDC1-AS.
4. the oxaliplatin and MDC1-AS prepared by a kind of preparation method as claimed in claim 3 transmit medicament carrier system altogether.
5. a kind of oxaliplatin as claimed in claim 4 and MDC1-AS transmit medicament carrier system targeted therapy cervical carcinoma altogether
Application.
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| 吕瑞等: "奥沙利铂对人***HeLa细胞株的体外作用", 《中山大学学报(医学科学版)》 * |
| 彭钊: "新型热敏磁性阳离子载药***共递送多柔比星及SATB1-shRNA靶向治疗胃癌实验研究", 《中国博士学位论文全文数据库 医药卫生科级辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113975244A (en) * | 2021-09-18 | 2022-01-28 | 上海交通大学医学院附属第九人民医院 | Bionic magnetic targeting cationic liposome and preparation method and application thereof |
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