CN107158409A - CD44 shRNA/PEG MZF NPs/DDP nano liposomes and preparation method thereof - Google Patents
CD44 shRNA/PEG MZF NPs/DDP nano liposomes and preparation method thereof Download PDFInfo
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- CN107158409A CN107158409A CN201710390020.8A CN201710390020A CN107158409A CN 107158409 A CN107158409 A CN 107158409A CN 201710390020 A CN201710390020 A CN 201710390020A CN 107158409 A CN107158409 A CN 107158409A
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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
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
- A61K48/0066—Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
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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
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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
- A61K9/127—Liposomes
Abstract
The invention belongs to medical science and gene engineering technology field, and in particular to a kind of CD44 shRNA/PEG MZF NPs/DDP nano liposomes and its preparation method and application.The nano liposomes are, using PEG MZF NPs magnetic nanospheres as carrier, to load DDP and plasmid CD44 shRNA.Its preparation method is first obtained PEG MZF NPS/DDP compounds, then be mixed to get the nano liposomes with CD44 shRNA plasmids.The invention also discloses application of the nano liposomes in treatment ovarian cancer is prepared.The nano liposomes of the present invention are under additional action of alternating magnetic field, there are preferable Proliferation Ability and apoptosis-induced effect to ovarian cancer cell, effect is substantially better than any single treatment, its mechanism may be by lowering the protein expression of CD44, Survivin, VEGF, Bcl 2, so as to suppress cell propagation, inducing cell apoptosis, suppress blood vessel hyperplasia or directly result in cell death.
Description
Technical field
The invention belongs to medical science and gene engineering technology field, and in particular to a kind of CD44-shRNA/PEG-MZF-NPs/
DDP nano liposomes and its preparation method and application.
Background technology
Oophoroma is one of most common tumour in female reproductive system, and grade malignancy is very high, and the death rate accounts for gynecological tumor
First place.Due to oophoroma in early symptom often not substantially, early detection is difficult, in being during patient assessment more than 70%
In late period, survival rate is only 25% within 5 years.The early diagnosis to oophoroma still lacks special effective method or means at present.Ovary
The treatment of cancer is main to be aided with chemotherapy after operative treatment at present, but conventional chemotherapy side effect is larger, and recurrence after operation rate and
The rate of transform is higher.Therefore, the new and effective early diagnosis means of oophoroma are explored and treatment method is significant.By multiple means
Organically combine and joint targeting diagnosis and treatment are carried out to oophoroma as important research direction.Gene therapy has turned into after tumour tradition
Another new treatment means, preferable application prospect is shown in therapeutic field of tumor after operation, radiotherapy, chemotherapy.Tumour heat
Treatment is to kill tumour cell by heat energy, not only can also strengthen the sensitiveness of chemicotherapy with independent utility.With nanometer skill
The development of art, the treatment that nanometer technology is applied into tumour is also of increasing concern., will if can be using magnetic Nano material as carrier
Gene therapy, chemotherapy, thermotherapy are organically combined, a kind of complementation collaboration, it is possible to form new tumor combined therapeutic method.
Adhesion molecule CD44 is a kind of transmembrane glycoprotein of cell membrane surface, is used as a kind of important hyaluronic acid receptor
(HA), with cell adherence, breed, break up and migrate relevant.CD44 unconventionality expression the generation of tumour, development, transfer and
Key player, especially ovarian cancer tissue are play in the assessment of prognosis, CD44 expression rates are up to 96.92%.Research shows：
CD44 expresses high ovarian cancer patients, and its clinical manifestation is heavier, and TNM stage is also higher, high-caliber CD44 expression and ovary
The prognosis mala of cancer patient is relevant, and CD44 can be used as ovarian cancer patients pathological diagnosis and the efficiency index of prognosis prediction.Therefore,
CD44 is likely to become the novel targets for the treatment of oophoroma.
Gene therapy is that allogenic gene or genetic fragment are transported in target cell, and what target gene was intervened controls
Treatment method.Current gene therapy method is applied in kinds of tumors Therapy study in succession, wherein RNAi (RNA
Interference RNA are disturbed) technology shows preferable application prospect in therapeutic field of tumor, the technology is to utilize double-strand
The RNA identifications of Dicer restriction endonucleases in the cell, with reference to producing the siRNA that active length is 21-23bp under, digestion
(siRNA) homologous mRNA in efficient specifically degradation of cell, blocks target gene, so as to produce gene silencing effect, suppresses
Target gene activity.Oophoroma targeted therapy is carried out using RNAi, preferable therapeutic effect is obtained.
Efficiently, it is the key for carrying out gene therapy stably to carry out gene transfer.Along with the development of nanometer technology, to receive
Rice grain is widely studied for gene transfer vector.Nano particle is easier to be modified, with good biocompatibility and compared with
Few immune response, it is easy to combined into tissue with cell surface specific receptor, or cell is entered by target cell phagocytosis
It is interior, the transhipment of gene is realized, and discharge in the cell, with higher gene transfering efficiency.Therefore, nano-carrier turns into great
The novel vehicle systems of application prospect.
Magnetic nanoparticle as nano material one kind, in addition to the function with general nano-carrier, due to its tool
There is special magnetic property, the magnetic sensitivities different with surrounding tissue are produced in the presence of externally-applied magnetic field, make T1 and T2 magnetic
The relaxation time of resonance image-forming changes, and can be applied to magnetic resonance imaging.Effect of the magnetic nanoparticle in alternating magnetic field
Under can produce fuel factor, heat energy can not only increase the function of chemotherapeutics, can also to tumour cell carry out target hyperthermia.Its
Middle metal oxide particle Fe3O4It is to apply more magnetic nano-particle.The gold such as manganese, zinc are added in nano oxidized iron construction
Category element can be prepared into various ferrites, wherein Mn0.6Zn0.4Fe2O4Not only there is carrier, the function of thermotherapy, also with automatic
The feature of temperature control and constant temperature.
Chemotherapy is clinically the most frequently used oncotherapy means, but most of chemotherapeutics can all produce it is serious
Toxic side effect, and chemotherapy is mainly effective to proliferation period cell, and single chemotherapy is difficult thoroughly to cure tumour.Cis-platinum (DDP) is
As one of clinical the most frequently used chemotherapeutics, the DNA replication dna of cancer cell can be suppressed, and damage structure on its cell membrane, have compared with
Strong broad spectrum anticancer effect, especially shows preferable curative effect in the treatment of oophoroma.But it is clinically used suitable at present
Platinum is to be injected intravenously, after intravenously administrable, to surrounding tissue, can make patient because plasma DD P concentration raises simultaneously rapid disperse quickly
There is symptom of digestive tract and bone marrow suppression, Toxicity of Kidney, neurotoxicity, the allergic reactions such as anorexia, nausea,vomiting,diarrhea
Etc. toxic side effect.Therefore, changing the administering mode and formulation of chemotherapeutics has important clinical meaning.Tumour is targetted
Administration, can not only effectively kill tumour cell, and can substantially reduce the dosage of chemotherapeutics, mitigate its toxic side effect.
Based on the characteristic of oophoroma CD44 gene high expressions, and magnetic nano particle target gene therapy, simple magnetic fluid
Thermotherapy, cisplatin chemotherapy all show good effect in terms of oncotherapy research, and prospect extensively, but acts on still aobvious single,
Under the guidance of tumor combined therapeutic principle, this research is contemplated：With Mn0.6Zn0.4Fe2O4Nanoparticle is used as gene and pharmaceutical carrier, profit
With the effect of its excellent magnetic responsiveness and effective heating temperature control, by CD44-shRNA gene therapies, DDP chemotherapy, magnetic current body heat
Treat and organically combine, have complementary advantages, it is possible to form a kind of new method for combining targeting diagnosis and treatment oophoroma.
The content of the invention
In order to solve existing gene therapy, problem present in chemotherapy, the present invention is provided a kind of to be modified with PEG
Mn0.6Zn0.4Fe2O4Nanoparticle (PEG-MZF-NPs) carries CD44-shRNA Antioncogenes and DDP chemotherapeutics for carrier
Nano liposomes and preparation method thereof, the nano liposomes can control CD44-shRNA genes under additional magnetic fields
Treat, DDP chemotherapy and Magnetic Fluid Hyperthermia are organically combined, have complementary advantages, targeted therapy is carried out to tumour, prior art is efficiently solved
The problem of.
The invention also discloses the preparation method of the CD44-shRNA/PEG-MZF-NPs/DDP nano liposomes, including
Following steps：
CD44-shRNA/PEG-MZF-NPS/DDP nano liposomes are prepared using Film-ultrasonic technique plus high-speed stirred：①
Prepare PEG-MZF-NPS/DDP compounds：Take a certain amount of PEG-MZF-NPS magnetic fluids to be mixed with appropriate DDP solution, add
Distilled water, ultrasonic disperse 15min, makes it fully react, filtering, and filtrate is PEG-MZF-NPS/DDP compounds, is placed on
It is kept in dark place in 4 DEG C of refrigerators standby；2. the 60 DEG C of water-baths of gelatin and PBS are taken after addition CD44-shRNA matter after Gelatin
Grain, is mixed；3. will 1. liquid and 2. liquid mixing, normal temperature, 2000r/min stirrings, until bottle wall film completely falls off hydration.Ultrasound,
Centrifugation, abandons supernatant and the non-encapsulated nano material of bottom of bottle, takes the brown property aqueous suspension as CD44-shRNA/PEG-MZF- in middle level
NPS/DDP nano liposomes.
Further, 1. middle PEG-MZF-NPS and DDP mass ratioes are 40 to step:1-80:PEG-MZF- behind 1, plus distilled water
The final concentration of 60 μ g/ml of NPS；Step 2. in by PEG-MZF-NPS and CD44-shRNA mass ratioes be 40:1 adds CD44-
shRNA。
It is that can discussion be carrier with PEG-MZF-NPS in the present invention, CD44-shRNA is transfected to tumour cell, detection
The biological characteristics that PEG-MZF-NPS is combined with CD44-shRNA.Agarose gel electrophoretogram is shown：Work as magnetic nanosphere
(PEG-MZF-NPs) it is 40 with the mass ratio of CD44-shRNA plasmids:When 1, PEG-MZF-NPs can be with all in articulated system
Plasmid, the ratio is best combination ratio, and carrier and the proportioning of plasmid amount provide foundation when being transfected for gene below.
Extracellular nuclease and intracellular lyase can degrade exogenous DNA.Safe efficient, controllable gene transfer
Method is the key for carrying out gene therapy.Magnetic Nano gene complex is observed in this experiment using DNase-I digestion experiments
Stability.As a result show：PEG-MZF-NPs of the time in 1-60min and CD44-shRNA plasmid composites, its electrophoresis pattern
In band brightness substantially do not change, keep stable, exposed CD44-shRNA plasmids to add after DNase-I digestion, about
Almost digestion completely, shows complex stabilities preferably, PEG-MZF-NPS can protect CD44-shRNA from nucleic acid after 1min
The digestion of enzyme.
In gene therapy, the selection of gene transfer vector is most important, as genophore, not only to have preferable group
Knit compatibility, in transportation can Protecting gene fragment be not degraded, reach target cell after can also effectively discharge.
Shown in the extracorporeal releasing experiment of this magnetic nanosphere (PEG-MZF-NPS) and CD44-shRNA plasmid composites, 1h, 4h,
In 8h, 12h, 24h, 2d, DNA burst sizes increase, the 3rd, the 4th day without significant difference, show that PEG-MZF-NPs can not only be protected
Plasmid, under suitable condition can also effectively released dna from degraded.
By carrier of PEG-MZF-NPS by after CD44-shRNA plasmid transfections to ovarian cancer cell, CD44 gene expressions are bright
Aobvious to decline, its transfection efficiency is 55.46 ± 4.50%, points out PEG-MZF-NPs to be applied to base as gene transfer vector
Because for the treatment of.
This experiment is differently intervened oophoroma HO8910 cells, and experiment is divided into seven groups, is respectively：It is negative
Control group；DDP groups；DDP/CD44-shRNA groups；MFH groups；CD44-shRNA/MFH groups；DDP/MFH groups；DDP/CD44-
ShRNA/MFH groups, calculate each group cell proliferation inhibition rate and apoptosis rate respectively.As a result DDP/CD44-shRNA/MFH is shown
The cell proliferation inhibition rate and apoptosis rate of group are obviously higher than remaining each group.Magnetic thermotherapy, DDP chemotherapy, CD44-shRNA genes are controlled
Cell propagation can effectively be suppressed by treating triple combination, and induce its tune to die, and effect is substantially better than any single therapy.
Western blot methods detection show PEG-MZF-NPS/DDP/CD44-shRNA/MFH groups in Survivin, VEGF,
The expression of Bcl-2 albumen is decreased obviously, and points out its cure mechanism may be with suppressing cell propagation, inducing cell apoptosis, suppression
Blood vessel hyperplasia processed or to directly result in cell death etc. relevant.
This PEG-MZF-NPS (Mn0.6Zn0.4Fe2O4) in magnetic Nano material external magnetic induction heating experiment, additional
Under the conditions of 235KHZ, 4KW, 35A high-frequency alternating magnetic field, the PEG-MZF-NPS of various concentrations can be brought rapidly up, 0~20min
Heating drastically, heats up gently after 20min, and keeps certain constant temperature, illustrates that the material has good heating constant temperature ability.Wherein
Concentration reaches 42 DEG C~43 DEG C for 60 μ g/ml PEG-MZF-NPS in 20min, and keeps stable, and the temperature is tumor thermotherapy
Ideal temperature.
The PEG-MZF-NPS that the present invention is used, with good dispersiveness, magnetic responsiveness and biocompatibility；Success is made
For PEG-MZF-NPS/DDP compounds, the compound and PEG-MZF-NPS equally have good magnetothermal effect, can be used for
The thermochemotherapy research of tumour；External magnetic resonance imaging shows that it has the application potential as magnetic resonance T2 relaxation contrast agents, has
The targeting diagnosis target integrated with treatment may be realized.
2nd, PEG-MZF-NPS is combined with CD44-shRNA, with good biological characteristics, and PEG-MZF-NPS can be protected
Protect digestion of the DNA from nuclease, and can under conditions of appropriate effectively released dna.Cell transfection assays confirmation, PEG-
CD44-shRNA Successful transfections to ovarian cancer cell, and restrained effectively the expression of CD44 genes by MZF-NPS, it is shown that
PEG-MZF-NPS can as gene transfer vector feasibility.
3rd, under additional action of alternating magnetic field, PEG-MZF-NPS/shRNA/DDP nano liposomes have to ovarian cancer cell
Preferable Proliferation Ability and apoptosis-induced effect, effect are substantially better than any single treatment, and its mechanism may be by lowering
CD44, Survivin, VEGF, Bcl-2 protein expression, thus suppress cell propagation, inducing cell apoptosis, suppress blood vessel hyperplasia or
Directly result in cell death.
Brief description of the drawings
Fig. 1 is the transmission electron microscope picture (TEM) of PEG-MZF-NPS/DDP compounds.
Fig. 2 is the infrared spectrogram of PEG-MZF-NPS and PEG-MZF-NPS/DDP compounds.
Fig. 3 is PEG-MZF-NPS heating curve figures in alternating magnetic field.
Fig. 4 is PEG-MZF-NPS/DDP compounds heating curve figure in alternating magnetic field.
Fig. 5 PEG-MZF-NPS external MRI imagings.
The PEG-MZF-NPS of Fig. 6 various concentrations T2 T2s r2.
The external MRI imagings of Fig. 7 PEG-MZF-NPS/DDP compounds.
Fig. 8 be different quality than PEG-MZF-NPS combined with CD44-shRNA plasmids after electrophoresis pattern；
Swimming lane 1：0:1 (i.e. 100ng DNAs, PEG-MZF-NPS is not added)；Swimming lane 2：5:1；Swimming lane 3：10:1；Swimming lane 4：
20:1；Swimming lane 5：40:1；Swimming lane 6：80:1；Marker：(Takara, DL5000, from top to bottom index zone be followed successively by 5000bp,
3000bp、2000bp、1500bp、1000bp、750bp、500bp、250bp、100bp)。
Fig. 9 is PEG-MZF-NPs and the digestion Protection electrophoresis pattern of CD44-shRNA plasmid composites
Swimming lane 1：100ng original plasmids DNALane；Swimming lane 2：1 hour；Swimming lane 3：4 hours；Swimming lane 4：8 hours；Swimming lane 5：
12 hours；Swimming lane 6：24 hours；Swimming lane 7：48 hours；Swimming lane 8：72 hours；Swimming lane 8：96 hours MARK：DNA Marker
(Takara, DL5000, from top to bottom index zone be followed successively by 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp,
500bp、250bp、100bp)。
Figure 10 is PEG-MZF-NPs magnetic Nano materials and the release experiment electrophoresis pattern of CD44-shRNA plasmid composites
Lane 1：100ng original plasmids DNA, Lane 2：Compound 1 minute, Lane 3：Compound 10 minutes, Lane
4：
Compound 30 minutes, Lane 5：Compound 45 minutes, Lane 6：Compound 1 hour.
The expression of CD44 gene mRNAs after Figure 11 oophoroma HO8910 cell transfecting CD44-shRNA plasmids.
The expression of CD44 albumen after Figure 12 oophoroma HO8910 cell transfecting CD44-shRNA plasmids
Oophoroma HO8910 after the magnetic induction of Figure 13 CD44-shRNA/PEG-MZF-NPS/DDP nano liposomes is heated
Survivin, VEGF, Bcl-2 protein expression situation in cell.
Embodiment
In order to manage understand the present invention, below in conjunction with the accompanying drawings, the present invention is further described for specific material, should not be understood
For the specific restriction to the present invention.Material employed in the present invention is as follows：
Embodiment：
The preparation of 1.PEG-MZF-NPS/DDP compounds and sign
The preparation of 1.1 PEG-MZF-NPS/DDP compounds
A certain amount of PEG-MZF-NPS magnetic fluids are taken to mix (PEG-MZF-NPS and DDP mass ratioes with appropriate DDP solution
40:1-80:1) distilled water (the final concentration of 60 μ g/ml of PEG-MZF-NPS), is added, ultrasonic disperse 15min makes it fully react,
Filtering, filtrate is PEG-MZF-NPS/DDP compounds, be placed in 4 DEG C of refrigerators be kept in dark place it is standby.
The Detection of Stability of 1.2 PEG-MZF-NPS/DDP compounds
PEG-MZF-NPS/DDP compounds are placed on 4 DEG C of refrigerator storages, the visible a small amount of tan precipitate of ttom of pipe after one week, warp
It can be resuspended after ultrasonic disperse 1min, illustrate that the compound has preferable suspension stability.
1.3 PEG-MZF-NPS/DDP compound transmission electron microscope observings
Take a small amount of PEG-MZF-NPS/DDP compounds, plus absolute ethyl alcohol ultrasonic disperse 15 minutes, drop has film copper mesh, natural
After drying, its pattern is observed under transmission electron microscope (TEM).Electron microscope is shown in Fig. 1, and the PEG-MZF-NPS/DDP compounds of preparation are put down
Equal particle diameter is 10nm or so, and size is more consistent.
Fourier infrared spectrum (FTIR) analysis of 1.4 PEG-MZF-NPS/DDP compounds
The change of PEG-MZF-NPS and PEG-MZF-NPS/DDP characteristic peaks is analyzed with Fourier infrared spectrum (FTIR).It is red
External spectrum figure is shown in Fig. 2, in the infrared spectrum of PEG-MZF-NPS/DDP compounds, in 530cm-1A new peak nearby is occurred in that,
For the Pt-N characteristic absorption peaks of cis-platinum, show that PEG-MZF-NPS successfully wraps up DDP.
Heating experiment of the 1.5 PEG-MZF-NPS and PEG-MZF-NPS/DDP compounds in alternating magnetic field
Compound concentration is 20 μ g/ml, 40 μ g/ml, 60 μ g/ml, 80 μ g/ml PEG-MZF-NPS magnetic fluids, is taken respectively
500 μ l are placed in flat based tubes, are inserted directly into test tube and (avoid contact to test tube wall) thermometer, start alternating magnetic field, ginseng
Number is set to 235KHZ, 4KW, 35A, a diameter of 3CM of magnetic induction coil, test tube bottom away from magnetic induction coil about 0.5cm, every
5min records a temperature, and using the time as abscissa, temperature is ordinate, draws the liter of PEG-MZF-NPS magnetic Nano materials
Warm curve.
PEG-MZF-NPS/DDP compounds 1ml is taken to add in flat based tubes, alternating magnetic field parameter is set to：235KHZ、
4KW, output current is respectively progress heating experiment under 15A, 25A, 35A, 45A, and detection method heats up real with PEG-MZF-NPS
Test, using the time as abscissa, temperature is that ordinate draws the compound heating curve.
Temperature-raising characteristic of the PEG-MZF-NPS and PEG-MZF-NPS/DDP compounds in alternating magnetic field is shown in Fig. 3, Fig. 4, two
Person can be warming up to 43 DEG C or so, and keep stable, the thermotherapy available for tumour.
External magnetic resonance imaging (MRI) research of 1.6 PEG-MZF-NPS and PEG-MZF-NPS/DDP compounds
It is 0.04 μ g/ml, 0.08 μ g/ml, 0.16 μ g/ml, 0.32 μ g/ml, 0.64 μ g/ml, 1.28 μ g/ml by concentration
PEG-MZF-NPS is placed in the ependoff pipes containing 1% agarose, and control group is the agarose for adding deionized water.Separately take
The μ l of PEG-MZF-NPS/DDP compounds 100 are placed in ependoff pipes.Using 7.0T Micro-MR scanners, internal diameter 3.0cm
Body coil, SE T2W1 sequences, parameter setting is as follows：Repetition time TR2500ms, echo time TE36ms, thickness 1.0mm,
FOV4cmx3.5cm, matrix 256 × 256.The corresponding T2 values of each sample are read, make r2 curves (R2=1/ under Origin afterwards
T2), it follows that the r2 relaxation rates of each concentration samples.
It is observed that with the increase of PEG-MZF-NPS concentration, its T2W1 signal is obvious from magnetic resonance imaging (Fig. 5)
Reduction, measures the T2 values of each concentration material, it follows that relaxation rate r2=6.06 (the μ g/ml of PEG-MZF-NPS materials-1s-1), see Fig. 6, point out PEG-MZF-NPS that there is the application potential as magnetic resonance T2 relaxation contrast agents.Fig. 7 is PEG-MZF-
The weighted image of NPS/DDP compounds, its signal is significantly lower than control group, shows that PEG-MZF-NPS equally may be used after being combined with DDP
To be used as contrast agent.
1.7 PEG-MZF-NPS and CD44-shRNA plasmid Binding experiments
By PEG-MZF-NPS and CD44-shRNA plasmids according to mass ratio 0:1、5:1、10:1、20:1、40:1、80:1 enters
Row mixing (the final concentration of 20ng/ μ l of DNA, μ l of cumulative volume 20 is supplied with ultra-pure water), room temperature is placed 30 minutes, with abundant
Reaction forms compound.Take 5 μ l compounds to enter row agarose gel electrophoresis (i.e. 100ng/ swimming lanes) respectively, check the situation of combination,
And filter out the best combination ratio of magnetic nanosphere and DNA.
The agarose gel electrophoretogram of magnetic nanosphere (PEG-MZF-NPs) and CD44-shRNA plasmid Binding experiments shows
Show, by 0:1、5:1、10:1、20:1、40:1 adds plasmid, the visible clearly band of swimming lane；From electrophoretogram, work as nano magnetic
Grain and CD44-shRNA mass ratio are 40:When 1, mass ratio 40 can be pointed out with plasmid all in articulated system substantially:1 is
PEG-MZF-NPs and CD44-shRNA combination optimal proportions, are shown in Fig. 8.
1.8 PEG-MZF-NPS digest Protection to the DNase-I of CD44-shRNA plasmids
By PEG-MZF-NPS and plasmid CD44-shRNA according to mass ratio 40:1 is mixed to form compound, adds Tango
Buffer (Thermo) and DNase-I enzymes, 37 DEG C of water-baths digest 1 minute, 10 minutes, 30 minutes, 45 minutes, 1 hour respectively,
Rapidly with isometric 100mmol/L EDTA solution terminating reactions.The plasmid in compound is eluted with SDS, phenol,
After chloroform, absolute ethyl alcohol precipitation, the washing of 75% ethanol are dried, it is dissolved in appropriate ultra-pure water, takes isometric product
Enter row agarose gel electrophoresis detection.Exposed DNA same method processing, is used as control.
In PEG-MZF-NPs and CD44-shRNA compound through DNase-I enzymic digestions 1-60min, its electrophoresis pattern
Band brightness does not substantially change, and keeps stable；Exposed CD44-shRNA plasmids are added after DNase-I enzymic digestions, about 1min
Almost digestion completely, band has been can't see on swimming lane afterwards, point out PEG-MZF-NPs can effectively protect the plasmid from
The digestion of DNase-I enzymes, is shown in Fig. 9.
1.9 PEG-MZF-NPS/CD44-shRNA DNA release experiments
By magnetic Nano material PEG-MZF-NPS and CD44-shRNA plasmids according to mass ratio 40:1 be mixed to form it is compound
Thing, the amount (10 μ g) of plasmid is mixed to 500ul with TE solution, to 37 DEG C of constant-temperature table 200rpm vibrations.Respectively at 1 hour, it is 4 small
When, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours 8 μ l of sampling, final each 5 μ l/ holes carry out Ago-Gel
Electrophoresis.
As a result show, in 1h, 4h, 8h, 12h, 24h, 2d, DNA burst sizes increase, the 3rd day, the 4th day without significant difference,
PEG-MZF-NPs can protect plasmid from degraded, and rational released dna, is shown in Figure 10 under suitable condition.
The inspection of CD44 gene expressions after 2.0 CD44-shRNA plasmids transfect oophoroma HO8910 cells through PEG-MZF-NPS
Survey
2.0.1 PEG-MZF-NPS infection protocols are by CD44-shRNA-EGFP plasmid transfections to HO8910 cells.
PEG-MZF-NPS is transfected：The day before transfection, by the nutrient solution of cell antibiotic-free containing 10%FBS, according to 1-4
×105/ hole density is inoculated in orifice plate, to ensure that cell density is up to 80% or so during transfection；During transfection, by the expression of same amount
Plasmid (CD44-shRNA-EGFP) and zero load pSilencer 3.1-H1neo plasmids are diluted in 100 μ l antibiotic-free serum-frees
In nutrient solution；In the nutrient solution that PEG-MZF-NPS is diluted in 100 μ l antibiotic-free serum-frees, 20min is stored at room temperature；Will be dilute
(PEG-MZF-NPS and DNA mass ratioes are 40 by DNA and PEG-MZF-NPS mixing after releasing:1) 30min, is stored at room temperature, shape is allowed to
Into compound.Therebetween, cell culture fluid to be transfected is changed into the fresh nutrient solution of antibiotic-free containing 10%FBS, 400 μ l/ holes；
The μ l of DNA and PEG-MZF-NPS compounds 200 are added per hole；Fully mix after, be placed in cell culture incubator continue cultivate 6h it
Afterwards, supernatant is abandoned, cell is washed with PBS 1 time, the fresh nutrient solution containing 10%FBS is added and continues to cultivate.
Flow cytomery PEG-MZF-NPS infection protocols are by the transfection of CD44-shRNA-EGFP plasmid transfections to cell
Efficiency be (55.46 ± 4.50) %, it was confirmed that PEG-MZF-NPs as gene transfer vector feasibility.
2.0.2 CD44-shRNA intervenes CD44 gene expression detections after HO8910 cells
After empty plasmid and CD44-shRNA plasmids 24h being transfected in oophoroma HO8910 cells through PEG-MZF-NPs,
After Real-time PCR methods detection display transfection CD44-shRNA plasmids, it can effectively suppress CD44 in HO8910 cells
MRNA expression (Figure 11).The detection of WesternBlot methods is shown after CD44-shRNA therapeutic interventions, in HO8910 cells
CD44 protein expression level significantly reduces (Figure 12), further demonstrate that CD44-shRNA can be transferred to by PEG-MZF-NPs
Ovarian cancer cell, and suppress the expression of CD44 genes.
The preparation of 2.1 PEG-MZF-NPS/DDP/CD44-shRNA nano liposomes
CD44-shRNA/PEG-MZF-NPS/DDP nano liposomes are prepared using Film-ultrasonic technique plus high-speed stirred：Take
A certain amount of PEG-MZF-NPS magnetic fluids mix (PEG-MZF-NPS and DDP mass ratioes 40 with appropriate DDP solution:1-80:
1) distilled water (the final concentration of 60 μ g/ml of PEG-MZF-NPS), is added, ultrasonic disperse 15min makes it fully react, filtered, filter
Liquid is PEG-MZF-NPS/DDP compounds, be placed in 4 DEG C of refrigerators be kept in dark place it is standby；2. gelatin and PBS are taken
60 DEG C of water-baths (are 40 by PEG-MZF-NPS and CD44-shRNA mass ratioes after adding CD44-shRNA plasmids after Gelatin:1
Add CD44-shRNA), mix；3. will 1. liquid and 2. liquid mixing, normal temperature, 2000r/min stirrings, until bottle wall film takes off completely
It is overboard to close.Ultrasound, centrifugation, abandon supernatant and the non-encapsulated nano material of bottom of bottle, take the brown property aqueous suspension as CD44- in middle level
ShRNA/PEG-MZF-NPS/DDP nano liposomes.
The propagation to HO8910 cells is heated in 2.2 CD44-shRNA/PEG-MZF-NPS/DDP nano liposomes magnetic induction
Suppress and apoptosis-induced effect
HO8910 cells are inoculated in the IMEM nutrient solutions containing 10% hyclone, be placed in 37 DEG C, saturated humidity, 5%
CO2Incubator in cultivate, every 2~3 days passage once, growth period cell experiment of taking the logarithm.Experiment packet：A groups are negative right
According to group；B groups are DDP groups；C groups are DDP/CD44-shRNA groups；D groups are MFH groups；E groups are CD44-shRNA/MFH groups；F groups are
DDP/MFH groups；G groups are CD44-shRNA/PEG-MZF-NPS/DDP nano liposomes magnetic induction heating groups (DDP/CD44-
ShRNA/MFH groups).B, c group are separately added into DDP, and c group cell transfecting CD44-shRNA plasmids, transfection method is the same；D groups are added
PEG-MZF-NPS magnetic fluids；E groups add PEG-MZF-NPS/CD44-shRNA (mass ratioes 40:1) compound；F groups add PEG-
MZF-NPS/DDP compounds；G groups add PEG-MZF-NPS/DDP/CD44-shRNA nano liposomes；MFH groups (d, e, f, g
Group) cell is put on SPG-10A-II high-frequency magnetic induction heating instrument plate coils and heats 1h, and non-MFH groups (a, b, c group) room temperature is put
1h is put, then continues each group cell in incubator to cultivate 48h, the inhibiting rate of MTT methods detection each group cell propagation, cell
Proliferation inhibition rate=(1- experimental groups OD values/control group OD values) × 100.Flow cytometry analysis Apoptosis situation.
As a result show, CD44-shRNA/PEG-MZF-NPS/DDP nano liposomes magnetic induction heating group cell inhibitory effects
Rate and apoptosis rate are obviously higher than other each groups (see Tables 1 and 2), it is shown that the hot triple combination of CD44-shRNA, DDP, magnetic controls
The clear superiority for the treatment of.
The CD44-shRNA/PEG-MZF-NPS/DDP nano liposomes magnetic induction of table 1 is heated to ovarian cancer cell HO8910
Inhibited proliferation
aP ＜ 0.001 are contrasted with negative control group；bP ＜ 0.001 are contrasted with DDP groups；cP ＜ 0.001 and CD44-shRNA groups
Contrast；dP ＜ 0.001 are contrasted with PEG-MZF-NPS/MFH groups；eP ＜ 0.001 are contrasted with CD44-shRNA/MFH groups；fP ＜
0.001 contrasts with DDP/MFH groups；gP ＜ 0.001 are contrasted with DDP/CD44shRNA/MFH groups,hP ＜ 0.05 and DDP/MFH groups pair
Than；iP ＜ 0.05 are contrasted with DDP/CD44shRNA/MFH groups
The CD44-shRNA/PEG-MZF-NPS/DDP nano liposomes magnetic induction of table 2 is heated to ovarian cancer cell HO8910
Apoptosis-induced effect
After 2.3 CD44-shRNA/PEG-MZF-NPS/DDP nano liposomes magnetic induction heating oophoroma HO8910 cells
The detection of correlative protein expression
Experiment packet：A groups are negative control group (control group)；B groups are CD44-shRNA/PEG-MZF-NPS/DDP nanometers
Liposome magnetic induction heating group (DDP/CD44-shRNA/MFH groups).A groups add physiological saline, put cellar culture in incubator
48h；B groups add PEG-MZF-NPS/DDP/CD44-shRNA nano liposomes, in SPG-10A-II high-frequency magnetic induction heating instruments
1h is heated on plate coil, cellar culture 48h in incubator is put.Western blot methods are detected in two groups of cells
The expression of Survivin, VEGF, BCl-2 albumen.Figure 13 is CD44-shRNA/PEG-MZF-NPS/DDP nano liposomes
Magnetic induction heat after in HO8910 cells Survivin, VEGF, Bcl-2 albumen testing result, GADPH is internal reference, with
Negative control group compares, and the protein expression level of DDP/CD44-shRNA/MFH groups is decreased obviously.
Claims (4)
1. a kind of CD44-shRNA/PEG-MZF-NPs/DDP nano liposomes, it is characterised in that with PEG-MZF-NPs nano magnetics
Grain is carrier, loads DDP and plasmid CD44-shRNA.
The preparation method of 2.CD44-shRNA/PEG-MZF-NPs/DDP nano liposomes, it is characterised in that comprise the following steps：
1. PEG-MZF-NPS/DDP compounds are prepared：A certain amount of PEG-MZF-NPS magnetic fluids are taken to be mixed with appropriate DDP solution
Close, add distilled water, ultrasonic disperse 15min makes it fully react, filter, filtrate is PEG-MZF-NPS/DDP compounds,
Be placed in 4 DEG C of refrigerators be kept in dark place it is standby；
2. take the 60 DEG C of water-baths of gelatin and PBS after adding CD44-shRNA plasmids after Gelatin, mix；
3. will 1. liquid and 2. liquid mixing, normal temperature, 2000r/min stirrings, until bottle wall film completely falls off hydration；Ultrasound, centrifugation,
Supernatant and the non-encapsulated nano material of bottom of bottle are abandoned, the brown property aqueous suspension as CD44-shRNA/PEG-MZF-NPS/DDP in middle level is taken
Nano liposomes.
3. method according to claim 2, it is characterised in that 1. middle PEG-MZF-NPS and DDP mass ratioes are 40 to step:
1-80:The final concentration of 60 μ g/ml of PEG-MZF-NPS behind 1, plus distilled water；Step 2. middle PEG-MZF-NPS and CD44-shRNA matter
Amount is than being 40:1.
4. the CD44-shRNA/PEG-MZF-NPs/DDP nano liposomes described in claim 1 are preparing treatment ovarian cancer
In application.
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| CN114949210A (en) * | 2022-05-20 | 2022-08-30 | 泰州市人民医院 | Preparation method and application of S2.2-PEG-MZF-NPs molecular probe |
| CN114949211A (en) * | 2022-05-20 | 2022-08-30 | 泰州市人民医院 | Preparation method and application of S2.2-PEG-MZF-NPs/DOX nanoliposome |
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