CN104758952B - Nano-carrier of medicine and gene and its production and use is delivered altogether - Google Patents
Nano-carrier of medicine and gene and its production and use is delivered altogether Download PDFInfo
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- CN104758952B CN104758952B CN201510103972.8A CN201510103972A CN104758952B CN 104758952 B CN104758952 B CN 104758952B CN 201510103972 A CN201510103972 A CN 201510103972A CN 104758952 B CN104758952 B CN 104758952B
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Abstract
The invention discloses a kind of nano-carrier of delivering medicine and gene and its production and use altogether；It is specifically related to a kind of while the common delivering nano-carrier TCPL siRNA PPX of load chemotherapeutics and genomic medicine, this delivery system is formed by polymeric prodrugs carrier TCPL, siRNA and multi-functional polyanionic polymer PPX by Electrostatic Absorption self assembly between composition；It can be achieved to be delivered to medicine and gene target into same tumour cell, and siRNA discharged in kytoplasm, the albumen of silence Bcl 2, promote apoptosis and release suppression of the Bcl 2 to Lonidamine；And the chemotherapeutics Lonidamine that mitochondria is acted on is delivered to mitochondria；The apoptosis of the two collaboration triggering mitochondria pathway, kills tumour cell jointly.The present invention passes through In vitro and in vivo activity evaluation, it was demonstrated that the system conveys each one-component better than simultaneously, can significantly improve its active anticancer, with clear and definite synergistic therapeutic effect.
Description
Technical field
The present invention relates to a kind of nano-carrier of delivering medicine and gene altogether, and in particular to a kind of load chemotherapeutics simultaneously
Common conveying with genomic medicine is classified targeting drug delivery system, and delivery system of the present invention being capable of targets neoplastic cells and delivery of gene medicine
Thing reaches cytoplasm and chemotherapeutics is delivered into mitochondria, the apoptosis of the two collaboration triggering mitochondria pathway, reaches collaboration
The purpose of tumour is treated, belongs to technical field of medicine.
Background technology
Cancer seriously threatens human health, and the treatment of single means typically is difficult to reach optimal curative effect.Two kinds or many
Plant therapy approach collaboration to work, be one of available strategy of oncotherapy.Chemotherapy, also referred to as chemotherapy, are current clinics
The Main Means for the treatment of.These antineoplastic majorities directly can quickly kill tumour cell, but its toxic side effect also makes
People is difficult to stand, and can also produce multidrug resistance using some chemotherapeutics more for a long time, and curative effect is controlled in further reduction
Really.Mitochondria, as the important organelle of mediating apoptosis, is the important therapy target of antineoplastic.But act on line
The curative effect of the chemotherapeutics of plastochondria clinically is general, and reason is due to that medicine reaches mitochondrial amount very little, and tumour is thin
Anti-apoptotic proteins on the mitochondria of born of the same parents suppress caused by the function of these medicines.Therefore, it will act on mitochondrial drug targeting
Mitochondria is delivered to, while eliminating the suppression of anti-apoptotic proteins using gene silent technology, chemotherapy and gene therapy can be achieved
Synergy, so as to strengthen antitumor curative effect, reduces toxic side effect.
RNA perturbation techniques (siRNA, siRNA) can specificity and optionally silencing of target genes expression.Its mechanism is
Exogenous double-stranded RNA such as siRNA enter after cell, and positive-sense strand and antisense are unwind into the presence of the DBPA in kytoplasm
Chain, subsequent antisense strand combines to form the silencing complex (RISC) of RNA inductions with some intracellular enzyme cuttings or unwindase etc. again.
The mRNA homologous regions specific binding of RISC and target gene, in binding site enzymolysis cutting mRNA, so that mRNA degradeds are led
Cause corresponding silenced gene expression.SiRNA is the double stranded RNA sequence of about 22 base pairs lengths, readily soluble in elecrtonegativity
Yu Shui, with efficient silence effect.However, this electronegative large biological molecules of siRNA are delivered in tumour cell relatively
Highly difficult, therefore, it is the key link for effectively realizing RNA silence functions to prepare suitable carrier.
At present, traditional chemotherapy and siRNA gene silencing combined treatment tumours, which have been obtained, greatly pays close attention to and obtains
Preferable therapeutic effect.It is used to deliver chemotherapeutics and therapeutic gene siRNA carrier altogether in recent years, including inorganic nano-particle,
Polymer micelle, lipid complex, dendrimer etc..These common delivery systems can realize the common load of medicine and gene, and
And can be simultaneously by chemotherapeutics and gene delivery to same tumour cell.However, can realize the two is more preferable in intracellular
Ground is delivered to respective action site, and it be maximally effective that such collaboration, which works,.Therefore, a kind of achievable tumour is designed thin
Born of the same parents target and intracellular action site targets and can load the delivery vector of medicine and gene simultaneously and will have great importance.
Polymer shell glycan-polyethyleneimine (CS-PEI, CP) is a kind of good non-viral gene of biocompatibility
Carrier material, can effectively protect siRNA, and can efficiently deliver siRNA and enter cell, realize that the expression of target gene is sunk
It is silent.Polyethyleneimine has much free primary amine in chitosan-polyethyleneimine, and these primary amine can not only combine electronegative
Genomic medicine, while can also be combined by chemical bond with antineoplastic chemotherapy medicine, so as to can realize that chemotherapeutics and gene are carried altogether.
The content of the invention
Purpose：In order to overcome the deficiencies in the prior art, the present invention provides receiving for a kind of delivering medicine altogether and gene
Meter Zai Ti, the delivery system is that the chemotherapeutics and gene that can be used for synergistic treatment tumour deliver administration nano-drug administration system altogether, is related to
A kind of nanometer for being self-assembly of common load medicine and gene delivery system altogether, self-assembled nanometer delivery system of the invention can be by
Chemotherapeutics and gene are delivered in target cell simultaneously, and gene is discharged in target cell cytoplasm, and chemotherapeutics is delivered to mitochondria；This
Chemotherapeutics and gene can cooperate with the apoptosis of triggering mitochondria pathway to treat tumour in delivery system.
Technical scheme：In order to solve the above technical problems, the technical solution adopted by the present invention is：
One kind delivers nano-carrier altogether, and compound is formed by polymeric prodrugs carrier TCPL and siRNA, then again with many work(
Energy polyanionic polymer PPX is self-assembly of common delivering nano-carrier TCPL-siRNA-PPX；
Wherein, polymeric prodrugs carrier TCPL chemical structural formula is as follows：Wherein, n, y, z are positive integer,
Multi-functional polyanionic polymer PPX chemical structural formula is as follows：
Wherein, p, m1、m2For positive integer, X is target ligand, selected from folic acid or lactobionic acid or RGD.
Above-mentioned common delivering nano-carrier, preparation method comprises the following steps：SiRNA solution is added under vortex
In the TCPL solution of volume, vortex 30s is stored at room temperature 30min；Then the PPX solution isometric with siRNA solution is taken in vortex
Lower to add, vortex 30s is stored at room temperature 30min, produces common delivering nano-carrier TCPL-siRNA-PPX.
Nano-carrier particle size range is 100nm-300nm, and current potential is -15mV-+5mV.It is applicable to intravenous injection administration.
Preferably, described common delivering nano-carrier, it is characterised in that：The siRNA is Bcl-2siRNA,
And/or；The multi-functional polyanionic polymer PPX is dissolved in pH 7.4 phosphate buffer formation PPX solution.
Above-mentioned common delivering nano-carrier is being prepared in treating cancer (particularly cervix cancer and liver cancer etc.) medicine
Purposes.
Present invention also offers a kind of polymeric prodrugs carrier TCPL, polymeric prodrugs carrier is TPP and Lonidamine point
It is not connected with PEI with amido link, PEI is connected with schiff bases key with oxidation chitosan again, and its chemical structural formula is as follows：
Wherein, n, y, z are positive integer.
The synthetic method of the polymeric prodrugs carrier TCPL is as follows：
Wherein, polyethyleneimine PEI molecular weight is 800-3500.
The preparation method of described polymeric prodrugs carrier, specifically includes following steps：
1) TPP-COOH synthesis：Triphenylphosphine TPP and 6- bromocaproic acid are with certain proportion mol ratio (1:1.0-3.0) throw
Material, is dissolved in anhydrous acetonitrile, the lower reaction 10-24h of nitrogen protection, is recrystallized to give TPP-COOH；
2) appropriate TPP-COOH is taken, anhydrous DMSO dissolvings add DCC and NHS (mol ratio DCC:NHS:TPP-COOH=
1.0-5.0:1.0-5.0:1), stirring reaction 6-24h at room temperature, is centrifuged off precipitation, supernatant is with the PEI's containing polyethyleneimine
Anhydrous DMSO solution mixing, stirring reaction 6-24h obtains reaction solution at room temperature；
3) appropriate amount of drug Lonidamine LND is taken, anhydrous DMSO dissolvings add DCC and NHS (mol ratio DCC:NHS:LND=
1.0-5.0:1.0-5.0:1), stirring reaction 6-24h at room temperature, is centrifuged off precipitating to obtain supernatant, supernatant and reaction solution
(TPP-COOH reacts with PEI) is well mixed, and continues stirring reaction 6-24h at room temperature, then reaction solution molecular weight cut-off value
1000 bag filter dialysis, is first dialysed with DMSO, then with different volumes ratio (DMSO:H2O volume ratios) the DMSO aqueous solution difference
Dialysis 1 time, each 24h；Finally dialysed with distilled water, dialyzate is freeze-dried to obtain TPP-PEI-LND；
4) appropriate TPP-PEI-LND is taken, is dissolved with DMSO；The acetate buffer (pH4.5) for aoxidizing chitosan adds dropwise
Enter into TPP-PEI-LND DMSO solution, then 4 DEG C of reaction 24-72h are dialysed with the bag filter of molecular weight cut-off value 3500,
Distilled water is dialysed, filtering with microporous membrane, and filtrate freeze-drying obtains polymeric prodrugs carrier TCPL.
Present invention also offers a kind of multi-functional polyanionic polymer PPX, it is characterized in that polyacrylic acid PAA and targeting are matched somebody with somebody
Base X is connected with polyethylene glycol PEG of the two ends with amino, and its chemical structural formula is as follows：
Wherein, p, m1, m2 are positive integer, and X is target ligand, selected from folic acid or lactobionic acid or RGD.
Described multi-functional polyanionic polymer PPX, synthetic route is as follows：
Wherein, polyacrylic acid PAA molecular weight is 2000-10000, and polyethylene glycol PEG molecular weight is 1000-6000.
The preparation method of the multi-functional polyanionic polymer PPX specifically includes following steps：
1) target ligand X 0.1mmol, are dissolved in Na2CO3In solution, 0.1-2mmol EDC, NHS are then respectively adding,
After stirring, activation 10-60min, H containing 0.1mmol is transferred to2N-PEG-NH2Na2CO3In solution, stirring is reacted at room temperature
1-12h；Dialysed using the bag filter of molecular weight cut-off value 1000, Na2CO3Solution is dialyzate, dialysis into outer liquid without X untill,
Again with distilled water dialysis 24h, PEG-X is obtained after freeze-drying；
2) appropriate polyacrylic acid is weighed, is dissolved in sodium carbonate liquor, polyacrylic acid carboxyl mole 4% is added
After EDC, NHS, 10-60min, PEG-X is added, 1-12h is reacted at room temperature, freeze-drying obtains multi-functional polyanion after dialysis
Polymer P PX, is kept in dark place.
Beneficial effect：The present invention is prepared into polymeric prodrugs TCPL by carrier of chitosan-polyethyleneimine, with elecrtonegativity
SiRNA is mixed, and the mixture is positively charged, then passes through electrostatic interaction and electronegative polymer poly acrylic acid-polyethylene glycol-target
The nano-carrier of common delivering medicine and gene is combined to form to aglucon X (PPX).Electronegative outer layer copolymer PPX, realizes that pH is quick
The modification of the multifunctions such as sense (PAA), long circulating (PEG) and active targeting (X).Meanwhile, the maskable kernel positive electricity of elecrtonegativity PPX
Lotus, reduction blood circulation when and plasma protein effect, reduce toxic side effect.Outer layer PAA is by after cellular uptake, in lysosome
Can acid-sensitive and kernel depart from, the schiff bases key between TCPL can be broken in acid condition, and PEI can be broken with proton sponge effect
Lysosome is split, the TCPL being broken in kytoplasm can promote siRNA release, and carrying medicine to reach mitochondria.
The nano-carrier of medicine and gene is carried altogether, because particle size range itself is 100-300nm, is applicable to intravenous injection
Administration.
Medicine and gene are loaded using chitosan-polyethyleneimine, carrier organism compatibility is good, degradable, safe nothing
Poison.
Outer layer elecrtonegativity anionic polymer make it that nano-carrier kernel positive charge during blood circulation is shielded, because
This reduces the effect with plasma protein, the possibility that reduction nano-carrier is recognized and removed by reticuloendothelial system.Outer layer anion
The modification of polymer can realize the functional modifications such as active targeting, improve accumulation of the nano-carrier in target cell, improve antitumor
Effect.After nano-carrier is absorbed by target cell, targeting line grain is realized in the releasable siRNA of intracellular, and using active targeting modification
The function of body, two kinds of therapy approach act synergistically on mitochondria, trigger the apoptosis of mitochondria pathway, have in oncotherapy effect
It is larger to improve.
Brief description of the drawings
Fig. 1 is the sign for the TCPL polymer that the present invention is prepared according to embodiment 2：(A) TCPL hydrogen spectrogram, (B) TCPL
Infrared spectrogram.
Fig. 2 is the hydrogen spectrogram for the PPX polymer that the present invention is prepared according to embodiment 4, and by taking PPX as an example, wherein F represents leaf
Acid.
Fig. 3 is sign of the present invention according to the self-assembled nanometer grain of embodiment 6：(A) TCPL and siRNA is with different quality ratio
The compound gel electrophoresis figure of preparation, the gel electrophoresis for the nanoparticle that (B) TCPL, siRNA and PPX is prepared with different mass ratioes
Figure, (C) TCPL/siRNA compounds (mass ratio 20/1) and TCPL/siRNA/PPX nanoparticles (mass ratio 20/1/2) particle diameter with
Potential diagram, transmission electron microscope picture (the Scale bar of (D) TCPL/siRNA/PPX nanoparticles (mass ratio 20/1/2):1μm).
Fig. 4 is intracellular transport process of the present invention according to the TCPL/siRNA/PPX nanoparticles of embodiment 7：(A) streaming
Cell instrument detection intake TCPL/siRNA/PPX nanoparticles HeLa cells (FITC mark TCPL, Cy3 mark siRNA, the two
It is same intracellular), mitochondria target head TPP delivery vectors reach mitochondria to the observation of (B) lazer scan confocal microscope in the cell
(FITC shows green fluorescence, and mitochondria dyes red with MitoTracker, and the two overlapping region is shown as yellow, Scale bar:
25 μm), the observation of (C) lazer scan confocal microscope separates (FITC after TCPL/siRNA/PPX nanoparticles enter cell in intracellular
Labeled vector shows green fluorescence, and Cy3 marks siRNA shows red fluorescence, Scale bar:25μm).
Fig. 5 is that the present invention breeds according to the extracorporeal suppression tumor cell of embodiment 8.
Fig. 6 is apoptosis result to HeLa cell of the present invention according to embodiment 9.
Fig. 7 is western of the present invention according to the mitochondria pathway apoptosis-related protein to HeLa cells of embodiment 10
Blotting is detected.
Fig. 8 is tumor tissues aspect graph of the present invention according to the internal antitumor drug effect of embodiment 11.
Embodiment
The present invention is further described below in conjunction with the accompanying drawings.
The present invention is realized by following technical scheme, is comprised the following steps that：
The synthetic schemes of TCPL polymeric prodrugs is specific as follows：Triphenylphosphine TPP and 6- bromocaproic acid reaction generation TPP-
COOH.Triphenylphosphine is introduced after carboxyl, using DCC and NHS activated carboxyls, is reacted in anhydrous DMSO with polyethyleneimine PEI
Generate TPP-PEI.Drug lonidamine (LND), its carboxyl is activated using DCC and NHS, anti-with TPP-PEI in anhydrous DMSO
TPP-PEI-LND should be generated.The acetate buffer (pH4.5) of oxidation chitosan is added dropwise to TPP-PEI-LND DMSO
In solution, reaction generation TCPL is freeze-dried, -20 DEG C of placements are standby after dialysis.
X is dissolved in Na2CO3In solution, the carboxyl added on EDC and NHS activation X, then with H2N-PEG-NH2Reaction with
Amido link is connected to form PEG-X.Equally, carboxyl of the polyacrylic acid with EDC and NHS activation thereon, with the PEG other ends on PEG-X
Amino react to form PAA-PEG-X (PPX), be kept in dark place.
Delivery system is fresh preparation to self-assembled nanometer altogether, prepares scheme specific as follows：By siRNA solution under vortex
It is added in isometric TCPL solution, vortex 30s is stored at room temperature 30min.Then isometric PPX solution is taken under vortex
Add, vortex 30s is stored at room temperature 30min, produce self-assembled nanometer delivery system TCPL-siRNA-PPX altogether.
Self assembly prepared by above-mentioned preparation method delivers the nano-carrier of medicine and gene, described nano-carrier grain altogether
Footpath scope is 100nm-300nm, and current potential is -15mV-+5mV.
Application of the nano-carrier of above-mentioned delivering medicine and gene altogether in treating cancer.
Embodiment 1
The synthetic schemes of TCPL polymeric prodrugs is specific as follows：Triphenylphosphine (TPP) and 6- bromocaproic acids are with mol ratio 1:
1.05 feed intake, and are dissolved in anhydrous acetonitrile, the lower reaction 16h of nitrogen protection, are recrystallized to give TPP-COOH.Take appropriate TPP-
COOH, anhydrous DMSO dissolvings, adds dicyclohexylcarbodiimide DCC and n-hydroxysuccinimide NHS (mol ratio DCC:NHS:
TPP-COOH=1.5:1.5:1), stirring reaction 12h at room temperature, is centrifuged off precipitation, supernatant and the anhydrous DMSO containing PEI are molten
Liquid is mixed, and stirring reaction 12h obtains reaction solution at room temperature.Appropriate LND is taken, anhydrous DMSO dissolvings add DCC and NHS (mol ratios
DCC:NHS:TPP-COOH=1.5:1.5:1), stirring reaction 12h at room temperature, is centrifuged off precipitation, supernatant and reaction solution
(TPP-COOH reacts with polyethyleneimine PEI) is well mixed, and continues stirring reaction 12h at room temperature, then reaction solution molecule
The bag filter dialysis of cutoff value 1000 is measured, is first dialysed 3 times with DMSO, each 12h；Again with different volumes ratio (DMSO:H2O volumes
Dialysed respectively 1 time than 80%, 50%, DMSO solution 20%), each 24h；Finally with distilled water dialysis 48h, liquid one is changed per 4h
It is secondary.Dialyzate is freeze-dried to obtain product TPP-PEI-LND.Appropriate TPP-PEI-LND is taken, is dissolved with DMSO；Shell is aoxidized to gather
The acetate buffer (pH4.5) of sugar is added dropwise in TPP-PEI-LND DMSO solution, 4 DEG C of reaction 48h, is then used and is divided
The bag filter dialysis of son amount cutoff value 3500, distilled water dialysis 48h, dialyzate 0.8 μm of filtering with microporous membrane, filtrate freezing
Polymeric prodrugs TCPL is dried to obtain, drying box is placed standby.
Embodiment 2
The Structural Identification of TCPL polymer.
TCPL polymer identifies structure by hydrogen nuclear magnetic resonance and infrared spectrum.Fig. 1 (A), hydrogen spectrum result is shown：TCPL
Hydrogen spectrum spectrogram on, chemical displacement value 7.75-7.91ppm be TPP benzene ring hydrogens characteristic peak, chemical displacement value is 2.27-
2.55ppm is the characteristic peak of the hydrogen of methylene on PEI, chemical displacement value be 5.84ppm be the hydrogen of methylene on LND feature
Peak, chemical displacement value is that 3.71-3.81ppm is the characteristic peak for aoxidizing the hydrogen of methylene on chitosan sugar chain, and features described above peak is equal
There is display, illustrate polymer TCPL synthesis success.
Fig. 1 (B), the results of FT-IR is shown：TCPL is in 1404cm-1There are C=N stretching vibration peaks, illustrate there is schiff bases key
Formation.
Embodiment 3
PPX Macroscopic single crystal schemes are specific as follows：By taking folic acid FA as an example.FA 0.1mmol are weighed, 0.1M is dissolved in
Na2CO3In solution, 0.2mmol EDC, NHS are then respectively adding, is stirred, after activation 30min, H containing 0.1mmol is transferred to2N-
PEG-NH20.1M Na2CO3In solution, 2h is reacted in stirring at room temperature.Dialysed using the bag filter of molecular weight cut-off value 1000,
0.01M Na2CO3Solution is dialyzate, dialysis into outer liquid without FA untill (ultraviolet detection), then with distilled water dialyse 24h.Freezing
PEG-FA is obtained after drying.Appropriate polyacrylic acid is weighed, is dissolved in sodium carbonate liquor, polyacrylic acid carboxyl mole is added
After 4% EDC, NHS, 30min, PEG-FA is added, 2h is reacted at room temperature, freeze-drying obtains PPX (PPF), lucifuge after dialysis
Preserve.
Embodiment 4
The Structural Identification of PPX polymer.
PPX polymer identifies structure by hydrogen nuclear magnetic resonance.Fig. 2, hydrogen spectrum result is shown：In PPX hydrogen spectrum spectrogram, change
Displacement study 8.67ppm is the characteristic peak of hydrogen on folic acid pteridine, and chemical shift 3.53ppm is the characteristic peak of hydrogen on PEG methylene, is changed
Displacement study 1.02-2.22ppm is the characteristic peak of methylene hydrogen on PAA, and there is display at features described above peak, illustrates polymer P PX's
Synthesize successfully.
Embodiment 5
The preparation of the common delivery system of TCPL/siRNA/PPX self-assembled nanometers.
Preparation scheme is specific as follows：SiRNA solution is added under vortex in isometric TCPL solution, vortex 30s,
It is stored at room temperature 30min；Then isometric PPX solution is taken to be added under vortex, vortex 30s is stored at room temperature 30min, produced.
Embodiment 6
Self-assembled nanometer grain is to the investigation of siRNA binding abilities and the sign of nanoparticle
SiRNA is compressed nanoparticle and protective capability is characterized by electrophoresis.TCPL/siRNA and TCPL/siRNA/PPX with
After different mass ratioes is combined, loading pigment is added, last volume is 10 μ L.It is added in 2% Ago-Gel, GelRed
Dyeing, with TAE buffer solutions as electrolyte, 40min is run under 50V.
Fig. 3 (A)-(D), the pattern of compound passes through transmission electron microscope observing.1 drop TCPL/siRNA/PPX nanoparticles are taken to drip to
On copper mesh, 10s is dyed with 1% uranyl acetate solution.Copper mesh is dried in 10min in electric Microscopic observation.
The size and surface charge of TCPL/siRNA/PPX nanoparticles are determined using dynamic light scattering.Particle diameter about 120nm,
Zeta electric potential about -8mV.
Embodiment 7
TCPL/siRNA/PPX nanoparticle HeLa cellular uptakes and intracellular transport process
Fig. 4 (A), cellular uptake uses flow cytometry analysis.HeLa cells are with every hole 1 × 105Cells/well is inoculated in 24
In orifice plate, 37 DEG C of culture 24h.According to nanoparticle preparation method prepare FITC-TCPL/Cy3-siRNA/PPX nanoparticles, with without
The culture mediums of RPMI 1640 of folic acid are adjusted to debita spissitudo.The culture medium in every hole is discarded, 1mL sample solution is added, continues to train
Support 4h.Sample solution is discarded afterwards, with cold PBS 3 times, is digested a moment with pancreatin per hole, is discarded pancreatin, add proper volume
PBS blow and beat into after cell suspension, 300 mesh nylon net filters, flow cytomery is carried out immediately.As a result nanoparticle energy is shown
Medicine and siRNA are delivered to same intracellular.
Fig. 4 (B)-(C), intracellular transport process uses confocal laser scanning microscope.HeLa cells with every hole 5 ×
104 cells are inoculated in laser co-focusing culture dish, 37 DEG C of culture 24h.The nanoparticle containing FITC-TCPL/siRNA/PPX
The culture mediums of RPMI 1640, when being added to culture 6h in culture dish, discard sample solution, add 100nM MitoTracker Red
Dye 30min.After cell is dyed, dyeing liquor is discarded, with cold PBS 2 times, 4% paraformaldehyde solution fixes 20min, enters
Row confocal laser scanning microscope.The culture mediums of RPMI 1640 of the nanoparticle containing FITC-TCPL/Cy3-siRNA/PPX, are added
The 6h into culture dish, discards sample solution, and with cold PBS 2 times, 4% paraformaldehyde solution fixes 20min, carries out laser and is total to
Focusing microscope is observed.As a result Intracellular drug targeted delivery is shown in mitochondria, and siRNA discharges in kytoplasm.
Embodiment 8
Cytotoxicity
Nanoparticle is analyzed using MTS methods and carries the influence of siRNA and Lonidamine to HeLa cell inhibitory effects.Growth conditions
Good HeLa cells are with 1 × 104Cells/well is added in 96 orifice plates, after incubated overnight, is discarded culture medium, is separately added into and contains
TCP/siSCR/PPX nanoparticles, TCP/siBcl-2/PPX nanoparticles, Lonidamine, CPL/siSCR/PPX nanoparticles, TCPL/
The culture medium of siSCR/PPX nanoparticles, TCPL/siBcl-2/PPX nanoparticles and TCPL/siBcl-2/PP nanoparticles etc., each
Sample at least 3 multiple holes, with normal cell as a control group, only plus culture medium for blank group.To there is each above-mentioned treatment group
Comparativity, fixed LND concentration is that 1.31 μ g/mL, siRNA concentration are 0.5 μ g/mL.Respectively after culture 24h and 72h, added per hole
20 μ L MTS solution, 37 DEG C of shaking 4h, ELIASA 490nm detection absorbances (A) of lucifuge.Fig. 5, cytotoxicity result shows,
Lonidamine and siBcl-2 can cooperate with the propagation for suppressing HeLa cells.
Embodiment 9
Apoptosis
The research of nanoparticle inducing cell apoptosis is entered according to the operation of AnnexinV-FITC cell apoptosis detection kits
OK.HeLa cells are with every hole 3 × 105Cell is inoculated in 6 orifice plates, after incubated overnight, is separately added into per hole containing TCP/siSCR/
PPX nanoparticles, TCP/siBcl-2/PPX nanoparticles, Lonidamine, CPL/siSCR/PPX nanoparticles, TCPL/siSCR/PPX receive
The grain of rice, TCPL/siBcl-2/PPX nanoparticles and TCPL/siBcl-2/PP nanoparticles etc..To make each above-mentioned treatment group have comparable
Property, fixed LND concentration is 1.31 μ g/mL, and siRNA concentration is 0.5 μ g/mL.Cultivate after 48h, PBS 2 times, added not per hole
The μ L of pancreatin 200 containing EDTA, add 1mL PBS and cell are collected into EP pipes after sucking, 2000rpm, 4 DEG C of centrifugation 5min.Abandon
Supernatant is removed, 500 μ L Binding buffer are added per hole and are resuspended.Added per hole after 5 μ L Annexin V-FITC mixings, then
5 μ L Propidium Iodide are added, lucifuge reaction 10min, and prepare corresponding single dye pipe at room temperature after mixing.Nylon membrane
After filtering, flow cytomery is carried out immediately.Fig. 6, apoptosis result shows that Lonidamine and siBcl-2 can cooperate with increase
The apoptosis rate of HeLa cells.
Embodiment 10
Western blotting detect the apoptosis-related protein of mitochondria pathway
After nanoparticle solution processing HeLa cells 48h, cell is collected in pancreatin digestion centrifugation.With appropriate cell pyrolysis liquid
Cell is resuspended in (containing 100 μ g/mL PMSF), and 1h, abundant cell lysis are placed on ice.4 DEG C, 12000rpm centrifugation 10min take
Clearly, protein content is detected using BCA methods.Protein sample is proportionally added into 6 × sample-loading buffer, and 100 DEG C of heating 5min make albumen
Denaturation, takes protein sample to be added in right amount in loading hole.Wet method transferring film after electrophoresis, contaminates film 1x Ponceauxs dye liquor after having turned
5min, 5% skim milk closing 1h.Plus primary antibody is incubated, primary antibody (such as Bcl-2, Bax, Caspase 3, Caspase 9) is used respectively
5% milk confining liquid dilution (1:500), stay overnight for 4 DEG C.Secondary antibody is incubated：Primary antibody is abandoned, PBS-T washes film 3 times, decolourize to shake at room temperature
Shaken on bed, each 10min.Film and 1% milk IgG/HRP (secondary antibody) are incubated together, 37 DEG C of shaking 2h.PBS- is used at room temperature
T, which is washed on decolorization swinging table after film, adds luminous nitrite ion, and room temperature effect 1min puts in gel imager and taken pictures.And use gel figure
As processing system scans OD value.Fig. 7, as a result shows, Lonidamine and siBcl-2 can cooperate with withering for triggering mitochondria pathway
Die.
Embodiment 11
Internal antitumor drug effect
H22 is inoculated in the right oxter of ICR male mices, treats tumour length to 100mm3When, 7 groups are randomly divided into, every group 6, is divided
Other tail vein injection administration.Experiment packet is respectively：Control group (physiological saline group), TCP/siSCR/PPX groups, LND groups
(1.05mg/kg), LND groups (40mg/kg), TCP/siBcl-2/PPX groups, TCPL/siSCR/PPX groups, TCPL/siBcl-2/
PPX groups.Dosage regimen：Tumor-bearing mice oxter knurl length is to 100mm3When start administration, be calculated as the 0th day with being administered for the first time, respectively
In 0, administration in 2,4,6,8,10,12 days.Dosage：SiRNA 0.2mg/kg, Lonidamine 1.05mg/kg.After treatment end,
Tumor tissues are taken out in dissection, are arranged according to every group, take pictures and weigh.Fig. 8, internal tumor suppression result show Lonidamine and
SiBcl-2 can cooperate with the growth for suppressing tumour.
Described above is only the preferred embodiment of the present invention, it should be pointed out that：For the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (9)
1. one kind delivers nano-carrier altogether, compound is formed by polymeric prodrugs carrier TCPL and siRNA, then again with it is multi-functional
Polyanionic polymer PPX is self-assembly of common delivering nano-carrier TCPL-siRNA-PPX；
Wherein, polymeric prodrugs carrier TCPL chemical structural formula is as follows：Wherein, n, y, z are positive integer,
Multi-functional polyanionic polymer PPX chemical structural formula is as follows：
Wherein, p, m1、m2For positive integer, X is target ligand, selected from folic acid or lactobionic acid or RGD.
2. according to claim 1 deliver nano-carrier altogether, preparation method comprises the following steps：By siRNA solution in whirlpool
Screw off and be added in isometric TCPL solution, vortex 30s is stored at room temperature 30min；Then take isometric with siRNA solution
PPX solution is added under vortex, and vortex 30s is stored at room temperature 30min, produces common delivering nano-carrier TCPL-siRNA-PPX.
3. according to claim 1 deliver nano-carrier altogether, it is characterised in that：The siRNA is Bcl-2siRNA, and/
Or；The multi-functional polyanionic polymer PPX is dissolved in pH 7.4 phosphate buffer formation PPX solution.
4. according to claim 1 deliver nano-carrier altogether, it is characterised in that：Multi-functional polyanionic polymer PPX's
Synthetic route is as follows：
Wherein, polyacrylic acid PAA molecular weight is 2000-10000, and polyethylene glycol PEG molecular weight is 1000-6000.
5. according to claim 1 deliver nano-carrier altogether, it is characterised in that：The multi-functional polyanionic polymer
PPX preparation method, specifically includes following steps：
1) target ligand X 0.1mmol, are dissolved in Na2CO3In solution, 0.1-2mmol EDC, NHS are then respectively adding, is stirred,
Activate after 10-60min, be transferred to H containing 0.1mmol2N-PEG-NH2Na2CO3In solution, 1-12h is reacted in stirring at room temperature；
Dialysed using the bag filter of molecular weight cut-off value 1000, Na2CO3Solution is dialyzate, dialysis into outer liquid without X untill, then with steaming
Distilled water dialysis 24h, PEG-X is obtained after freeze-drying；
2) appropriate polyacrylic acid is weighed, is dissolved in sodium carbonate liquor, the EDC of addition polyacrylic acid carboxyl mole 4%,
After NHS, 10-60min, PEG-X is added, 1-12h is reacted at room temperature, freeze-drying obtains multi-functional polyanion polymerization after dialysis
Thing PPX, is kept in dark place.
6. use of the common delivering nano-carrier in preparing for treating cancer medicine according to claim any one of 1-5
On the way.
7. a kind of polymeric prodrugs carrier TCPL, it is characterised in that：Polymeric prodrugs carrier be TPP and Lonidamine respectively with
PEI is connected with amido link, and PEI is connected with schiff bases key with oxidation chitosan again, and its chemical structural formula is as follows：
Wherein, n, y, z are positive integer.
8. polymeric prodrugs carrier TCPL according to claim 7, it is characterised in that：The polymeric prodrugs carrier TCPL
Synthetic method it is as follows：
Wherein, polyethyleneimine PEI molecular weight is 800-3500.
9. the polymeric prodrugs carrier TCPL according to claim 7 or 8, it is characterised in that：The polymeric prodrugs carrier
TCPL preparation method, specifically includes following steps：
1) TPP-COOH synthesis：Triphenylphosphine TPP and 6- bromocaproic acid are fed intake with certain proportion mol ratio, are dissolved in anhydrous acetonitrile
In, the lower reaction 10-24h of nitrogen protection is recrystallized to give TPP-COOH；
2) appropriate TPP-COOH is taken, anhydrous DMSO dissolvings add DCC and NHS, stirring reaction 6-24h, is centrifuged off sinking at room temperature
Form sediment, supernatant is mixed with the anhydrous DMSO solution of the PEI containing polyethyleneimine, and stirring reaction 6-24h obtains reaction solution at room temperature；
3) appropriate amount of drug Lonidamine LND is taken, anhydrous DMSO dissolvings add DCC and NHS, at room temperature stirring reaction 6-24h, from
The heart is removed and precipitates to obtain supernatant, and supernatant is well mixed with reaction solution, continues stirring reaction 6-24h at room temperature, then reaction solution
Dialysed, first dialysed with DMSO with the bag filter of molecular weight cut-off value 1000, then with different volumes than DMSO aqueous solution difference it is saturating
Analysis 1 time, each 24h；Finally dialysed with distilled water, dialyzate is freeze-dried to obtain TPP-PEI-LND；
4) appropriate TPP-PEI-LND is taken, is dissolved with DMSO；The acetate buffer of oxidation chitosan is added dropwise to TPP-PEI-
In LND DMSO solution, then 4 DEG C of reaction 24-72h are dialysed with the bag filter of molecular weight cut-off value 3500, distilled water dialysis,
Filtering with microporous membrane, filtrate freeze-drying obtains polymeric prodrugs carrier TCPL.
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| CN107261156B (en) * | 2017-06-07 | 2021-03-09 | 华侨大学 | SANH-based MSR-1 magnetosome compound carrying siRNA and adriamycin together and preparation method thereof |
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