CN104758952A - Co-delivery nano-carrier of drug and gene, preparation method and application thereof - Google Patents

Co-delivery nano-carrier of drug and gene, preparation method and application thereof Download PDF

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CN104758952A
CN104758952A CN201510103972.8A CN201510103972A CN104758952A CN 104758952 A CN104758952 A CN 104758952A CN 201510103972 A CN201510103972 A CN 201510103972A CN 104758952 A CN104758952 A CN 104758952A
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carrier
tcpl
ppx
sirna
solution
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CN104758952B (en
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姜虎林
张兵锋
邢磊
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China Pharmaceutical University
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G81/00—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers
    • C08G81/02—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers at least one of the polymers being obtained by reactions involving only carbon-to-carbon unsaturated bonds
    • C08G81/024—Block or graft polymers containing sequences of polymers of C08C or C08F and of polymers of C08G
    • C08G81/025—Block or graft polymers containing sequences of polymers of C08C or C08F and of polymers of C08G containing polyether sequences
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A61K31/416—1,2-Diazoles condensed with carbocyclic ring systems, e.g. indazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
    • C08G65/33396—Polymers modified by chemical after-treatment with organic compounds containing nitrogen having oxygen in addition to nitrogen
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
    • C08G73/02—Polyamines
    • C08G73/0206—Polyalkylene(poly)amines
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G81/00—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers

Abstract

The invention discloses a co-delivery nano-carrier of drugs and genes, a preparation method and an application thereof; and particularly relates to a co-delivery nano-carrier TCPL-siRNA-PPX which can carry a chemotherapeutic drug and a genetic drug simultaneously. The drug delivery system is composed of a polymer prodrug carrier TCPL, siRNA and a multifunctional polyanionic polyer PPX through electrostatic adsorption among the components in a self-assembly manner. The co-delivery nano-carrier can achieve targetedly delivery of the drug and the gene to the same tumor cell, release the siRNA at the cytoplasm, silence Bcl-2 protein, promote apotosis and relieve inhibition of the Bcl-2 on lonidamine, and can deliver the chemotherapeutic drug, lonidamine, which is mitochondrion-acted to mitochondria so that the chemotherapeutic drug and the gene can synergistically trigger the apotosis of a mitochondrial pathway and kill tumor cells cooperatively. Through in-vivo and in-vitro activity evaluation, the drug delivery system is proved to be better than drug delivery carriers which deliver single components respectively at the same time, can significantly improve the anti-cancer activity of the drug and the gene and has a definite synergistic treatment effect.

Description

Be total to nano-carrier of delivering drugs and gene and its production and use
Technical field
The present invention relates to a kind of nano-carrier of delivering drugs and gene altogether, the common conveying classification targeting drug delivery system of load chemotherapeutics and genomic medicine while of being specifically related to a kind of, delivery system of the present invention targets neoplastic cells delivery of gene medicine can arrive Cytoplasm and chemotherapeutics is delivered to mitochondrion, the two collaborative apoptosis triggering mitochondria pathway, reach the object of Synergistic treatment tumor, belong to technical field of medicine.
Background technology
Human health in cancer serious threat, and the treatment of single means is generally difficult to reach best curative effect.Two or more therapy approach work in coordination with onset, are one of available strategies of oncotherapy.Chemotherapy, also claims chemotherapy, is the Main Means of current clinical treatment.These antitumor drug majorities directly can kill tumor cell fast, but its toxic and side effects also makes us being difficult to stand, and more importantly use some chemotherapeutics also can produce multidrug resistance for a long time, reduce therapeutic effect further.Mitochondrion, as the important organelle of mediating apoptosis, is the important therapy target of antitumor drug.But it is general to act on mitochondrial chemotherapeutics curative effect clinically, reason is because medicine arrives mitochondrial amount very little, and the anti-apoptotic proteins on the mitochondrion of tumor cell suppresses caused by the function of these medicines.Therefore, mitochondrial drug targeting will be acted on and be delivered to mitochondrion, and adopt gene silent technology to eliminate the suppression of anti-apoptotic proteins simultaneously, the synergism of chemotherapy and gene therapy can be realized, thus strengthen antitumor curative effect, reduce toxic and side effects.
RNA perturbation technique (siRNA, siRNA) can specificity and optionally silencing of target genes expression.Its mechanism is exogenous double-stranded RNA as after siRNA enters cell, unwind into positive-sense strand and antisense strand under the effect of the DBPA in kytoplasm, antisense strand combines with more intracellular enzyme or the unwindase etc. cut the silencing complex (RISC) forming RNA and induce again subsequently.The mRNA homology region specific binding of RISC and target gene, at binding site enzymolysis cutting mRNA, thus makes mRNA degraded cause corresponding silenced gene expression.SiRNA is the double stranded RNA sequence of about 22 base pairs length, in elecrtonegativity, soluble in water, has efficient reticent effect.But this electronegative biomacromolecule of siRNA is delivered in tumor cell relatively very difficult, therefore, preparing suitable carrier is the key link effectively realizing the reticent function of RNA.
At present, traditional chemotherapy and siRNA gene silencing combined treatment tumor have obtained to be paid close attention to and obtains good therapeutic effect greatly.In recent years for sending the carrier of chemotherapeutics and therapeutic gene siRNA altogether, comprise inorganic nano-particle, polymer micelle, lipid complex, dendrimer etc.These common delivery systems can realize carrying altogether of medicine and gene, and can simultaneously by chemotherapeutics and gene delivery in same tumor cell.But can the two be realized in born of the same parents to be delivered to respective action site better, so collaborative onset should be the most effective.Therefore, design and a kind ofly realize action site targeting in tumor cell targeting and born of the same parents and can the delivery vector of drug loading and gene simultaneously will have great importance.
Polymer shell polysaccharide-polymine (CS-PEI, CP) be a kind of biocompatibility good non-viral gene carrier material, effectively can protect siRNA, and siRNA can be sent efficiently and enter cell, the expression silencing of realize target gene.In chitosan-polymine, polymine has a lot of free primary amine, and these primary amine in conjunction with electronegative genomic medicine, also can not only be combined with antineoplastic chemotherapy medicine by chemical bond simultaneously, thus can realize chemotherapeutics and gene carries altogether.
Summary of the invention
Object: in order to overcome the deficiencies in the prior art, the invention provides a kind of nano-carrier of delivering drugs and gene altogether, this delivery system can be used for the chemotherapeutics of Synergistic treatment tumor and gene sends administration nano-drug administration system altogether, relate to the nanometer delivery system altogether that a kind of self assembly forms common medicine carrying thing and gene, chemotherapeutics and gene can be delivered in target cell by self-assembled nanometer delivery system of the present invention simultaneously, at target cell cytoplasm release gene, chemotherapeutics is delivered to mitochondrion; In this delivery system, chemotherapeutics and gene can be worked in coordination with the apoptosis triggering mitochondria pathway and be treated tumor.
Technical scheme: for solving the problems of the technologies described above, the technical solution used in the present invention is:
One sends nano-carrier altogether, forms complex by polymeric prodrugs carrier TCPL and siRNA, and then is formed with multi-functional polyanionic polymer PPX self assembly and send nano-carrier TCPL-siRNA-PPX altogether;
Wherein, the chemical structural formula of polymeric prodrugs carrier TCPL is as follows: wherein, and n, y, z are positive integer,
The chemical structural formula of multi-functional polyanionic polymer PPX is as follows:
Wherein, p, m 1, m 2for positive integer, X is target ligand, is selected from folic acid or lactobionic acid or RGD.
Above-mentioned sends nano-carrier altogether, and preparation method comprises the following steps: joined by siRNA solution in isopyknic TCPL solution under vortex, vortex 30s, room temperature leaves standstill 30min; Then get isopyknic PPX solution with siRNA solution to add under vortex, vortex 30s, room temperature leaves standstill 30min, obtains and sends nano-carrier TCPL-siRNA-PPX altogether.
Nano-carrier particle size range is 100nm-300nm, and current potential is-15mV-+5mV.Be applicable to intravenous administration.
Preferably, described sends nano-carrier altogether, it is characterized in that: described siRNA is Bcl-2siRNA, and/or; The phosphate buffer that described multi-functional polyanionic polymer PPX is dissolved in pH 7.4 forms PPX solution.
Above-mentioned nano-carrier of sending altogether is for the preparation of the purposes in Therapeutic cancer (particularly cervical cancer and hepatocarcinoma etc.) medicine.
Present invention also offers a kind of polymeric prodrugs carrier TCPL, polymeric prodrugs carrier is that TPP is connected with amido link with PEI respectively with lonidamine, and PEI is connected with oxidation chitosan with Schiff's base key again, and its chemical structural formula is as follows:
Wherein, n, y, z are positive integer.
The synthetic method of described polymeric prodrugs carrier TCPL is as follows:
Wherein, the molecular weight of polymine PEI is 800-3500.
The preparation method of described polymeric prodrugs carrier, specifically comprises the following steps:
1) synthesis of TPP-COOH: triphenylphosphine TPP and 6-bromocaproic acid feed intake with certain proportion mol ratio (1:1.0-3.0), and be dissolved in anhydrous acetonitrile, react 10-24h under nitrogen protection, recrystallization obtains TPP-COOH;
2) appropriate TPP-COOH is got, anhydrous DMSO dissolves, add DCC and NHS (mol ratio DCC:NHS:TPP-COOH=1.0-5.0:1.0-5.0:1), stirred at ambient temperature reaction 6-24h, centrifugal removing precipitation, supernatant mixes with the anhydrous DMSO solution containing polymine PEI, and stirred at ambient temperature reaction 6-24h obtains reactant liquor;
3) appropriate amount of drug lonidamine LND is got, anhydrous DMSO dissolves, add DCC and NHS (mol ratio DCC:NHS:LND=1.0-5.0:1.0-5.0:1), stirred at ambient temperature reaction 6-24h, centrifugal removing precipitates to obtain supernatant, supernatant is mixed homogeneously with reactant liquor (TPP-COOH and PEI reacts), stirring reaction 6-24h is continued under room temperature, then the reactant liquor bag filter of molecular weight cut-off value 1000 is dialysed, first dialyse with DMSO, dialyse respectively 1 time with the DMSO aqueous solution of different volumes ratio (DMSO:H2O volume ratio) again, each 24h; Finally use distill water dialysis, dialysis solution obtains TPP-PEI-LND through lyophilization;
4) get appropriate TPP-PEI-LND, dissolve with DMSO; The acetate buffer (pH4.5) of oxidation chitosan dropwise joins in the DMSO solution of TPP-PEI-LND, 4 DEG C of reaction 24-72h, then dialyse with the bag filter of molecular weight cut-off value 3500, distill water dialysis, filtering with microporous membrane, filtrate lyophilization 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 is connected with the Polyethylene Glycol PEG of two end band amino with target ligand X, its chemical structural formula is as follows:
Wherein, p, m1, m2 are positive integer, and X is target ligand, are 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 described multi-functional polyanionic polymer PPX specifically comprises the following steps:
1) target ligand X 0.1mmol, is dissolved in Na 2cO 3in solution, then add 0.1-2mmol EDC, NHS respectively, stir, after activation 10-60min, be transferred to containing 0.1mmol H 2n-PEG-NH 2na 2cO 3in solution, stir, under room temperature, react 1-12h; Adopt the bag filter dialysis of molecular weight cut-off value 1000, Na 2cO 3solution is dialysis solution, dialyses without X to outer liquid, then uses distill water dialysis 24h, obtain PEG-X after lyophilization;
2) take appropriate polyacrylic acid, be dissolved in sodium carbonate liquor, add EDC, NHS of polyacrylic acid carboxyl mole 4%, after 10-60min, add PEG-X, under room temperature, react 1-12h, dialysis postlyophilization obtains multi-functional polyanionic polymer PPX, keeps in Dark Place.
Beneficial effect: the present invention with chitosan-polymine for carrier is prepared into polymeric prodrugs TCPL, mix with elecrtonegativity siRNA, this mixture strip positive charge, then the nano-carrier forming delivering drugs and gene is altogether combined by electrostatic interaction and electronegative polymer poly acrylic acid-Polyethylene Glycol-target ligand X (PPX).Electronegative outer layer copolymer PPX, realizes the multifunctions such as pH sensitivity (PAA), long circulating (PEG) and active targeting (X) and modifies.Meanwhile, elecrtonegativity PPX maskable kernel positive charge, with the effect of plasma protein when reducing blood circulation, reduction toxic and side effects.Outer PAA is by after cellular uptake, can acid-sensitive and kernel depart from lysosome, the Schiff's base key between TCPL can rupture in acid condition, and PEI can proton sponge effect to break lysosome, can promote the release of siRNA at the TCPL of kytoplasm Fracture, and carrying medicine arrives mitochondrion.
The nano-carrier of medicine carrying thing and gene, because particle size range own is 100-300nm, is applicable to intravenous administration altogether.
Adopt chitosan-polymine drug loading and gene, the carrier organism compatibility is good, degradable, safety non-toxic.
Outer elecrtonegativity anionic polymer makes nano-carrier kernel positive charge conductively-closed in blood circulation process, therefore reduces the effect with plasma protein, reduce nano-carrier by reticuloendothelial system identification and removing may.The modification of outer anionic polymer can realize the functional modifications such as active targeting, improves the accumulation of nano-carrier target cell, improves antitumous effect.After nano-carrier is absorbed by target cell, can discharge siRNA in born of the same parents, and utilize active targeting modification to realize the mitochondrial function of targeting, two kinds of therapy approach act synergistically on mitochondrion, trigger the apoptosis of mitochondria pathway, oncotherapy effect improves a lot.
Accompanying drawing explanation
Fig. 1 is the sign of the TCPL polymer that the present invention is prepared according to embodiment 2: the hydrogen spectrogram of (A) TCPL, the infrared spectrogram of (B) TCPL.
Fig. 2 is the hydrogen spectrogram of the PPX polymer that the present invention is prepared according to embodiment 4, and for PPX, wherein F represents folic acid.
Fig. 3 is the sign of the present invention according to the self-assembled nanometer grain of embodiment 6: (A) TCPL and siRNA is with the complex gel electrophoresis figure of different quality than preparation, (B) gel electrophoresis figure of nanoparticle prepared with different mass ratioes of TCPL, siRNA and PPX, (C) TCPL/siRNA complex (mass ratio 20/1) and TCPL/siRNA/PPX nanoparticle (mass ratio 20/1/2) particle diameter and potential diagram, the transmission electron microscope picture (Scale bar:1 μm) of (D) TCPL/siRNA/PPX nanoparticle (mass ratio 20/1/2).
Fig. 4 is the intracellular transport process of the present invention according to the TCPL/siRNA/PPX nanoparticle of embodiment 7: HeLa cell (the FITC labelling TCPL of (A) flow cytomery picked-up TCPL/siRNA/PPX nanoparticle, Cy3 labelling siRNA, the two is in same cell), (B) at intracellular mitochondrial target head TPP delivery vector arrival mitochondrion, (FITC shows green fluorescence in lazer scan confocal microscope observation, mitochondrion MitoTracker dyes redness, the two overlapping region is shown as yellow, Scale bar:25 μm), (C) lazer scan confocal microscope is observed after TCPL/siRNA/PPX nanoparticle enters cell, is separated (FITC labeled vector display green fluorescence in born of the same parents, Cy3 labelling siRNA shows red fluorescence, Scale bar:25 μm).
Fig. 5 is that the present invention breeds according to the extracorporeal suppression tumor cell of embodiment 8.
Fig. 6 is the apoptosis result to HeLa cell of the present invention according to embodiment 9.
Fig. 7 is that the present invention detects according to the westernblotting of the mitochondria pathway apoptosis-related protein to HeLa cell of embodiment 10.
Fig. 8 is the tumor tissues aspect graph of the present invention according to the anti-tumor in vivo drug effect of embodiment 11.
Detailed description of the invention
Below in conjunction with accompanying drawing, the present invention is further described.
The present invention is realized by following technical scheme, and concrete steps are as follows:
The synthetic schemes of TCPL polymeric prodrugs is specific as follows: triphenylphosphine TPP and the reaction of 6-bromocaproic acid generate TPP-COOH.After triphenylphosphine introduces carboxyl, adopt DCC and NHS activated carboxyl, react with polymine PEI in anhydrous DMSO and generate TPP-PEI.Drug lonidamine (LND), adopts DCC and NHS to activate its carboxyl, reacts generate TPP-PEI-LND in anhydrous DMSO with TPP-PEI.The acetate buffer (pH4.5) of oxidation chitosan dropwise joins in the DMSO solution of TPP-PEI-LND, and reaction generates TCPL, and dialysis postlyophilization ,-20 DEG C of placements are for subsequent use.
X is dissolved in Na 2cO 3in solution, add the carboxyl on EDC and NHS activation X, then with H 2n-PEG-NH 2reaction is connected to form PEG-X with amido link.Equally, polyacrylic acid EDC and NHS activates the carboxyl on it, reacts and forms PAA-PEG-X (PPX), keep in Dark Place with the amino of the PEG other end on PEG-X.
Self-assembled nanometer altogether delivery system is all fresh preparations, and preparation scheme is specific as follows: joined under vortex in isopyknic TCPL solution by siRNA solution, vortex 30s, room temperature leaves standstill 30min.Then get isopyknic PPX solution to add under vortex, vortex 30s, room temperature leaves standstill 30min, obtains self-assembled nanometer delivery system TCPL-siRNA-PPX altogether.
Self assembly prepared by above-mentioned preparation method is total to the nano-carrier of delivering drugs and gene, and described nano-carrier particle size range is 100nm-300nm, and current potential is-15mV-+5mV.
The application of nano-carrier on Therapeutic cancer of above-mentioned delivering drugs and gene altogether.
Embodiment 1
The synthetic schemes of TCPL polymeric prodrugs is specific as follows: triphenylphosphine (TPP) and 6-bromocaproic acid feed intake with mol ratio 1:1.05, are dissolved in anhydrous acetonitrile, and react 16h under nitrogen protection, recrystallization obtains TPP-COOH.Get appropriate TPP-COOH, anhydrous DMSO dissolves, add dicyclohexylcarbodiimide DCC and N-hydroxy-succinamide NHS (mol ratio DCC:NHS:TPP-COOH=1.5:1.5:1), stirred at ambient temperature reaction 12h, centrifugal removing precipitation, supernatant mixes with the anhydrous DMSO solution containing PEI, and stirred at ambient temperature reaction 12h obtains reactant liquor.Get appropriate LND, anhydrous DMSO dissolves, add DCC and NHS (mol ratio DCC:NHS:TPP-COOH=1.5:1.5:1), stirred at ambient temperature reaction 12h, centrifugal removing precipitation, supernatant is mixed homogeneously with reactant liquor (TPP-COOH reacts with polymine PEI), stirring reaction 12h is continued under room temperature, then the reactant liquor bag filter of molecular weight cut-off value 1000 is dialysed, and first dialyses 3 times with DMSO, each 12h; Dialyse respectively 1 time with the DMSO solution of different volumes ratio (DMSO:H2O volume ratio 80%, 50%, 20%) again, each 24h; Finally use distill water dialysis 48h, every 4h changes liquid once.Dialysis solution obtains product TPP-PEI-LND through lyophilization.Get appropriate TPP-PEI-LND, dissolve with DMSO; The acetate buffer (pH4.5) of oxidation chitosan dropwise joins in the DMSO solution of TPP-PEI-LND, 4 DEG C of reaction 48h, then dialyse with the bag filter of molecular weight cut-off value 3500, distill water dialysis 48h, the dialysis solution filtering with microporous membrane of 0.8 μm, filtrate lyophilization obtains polymeric prodrugs TCPL, and drying baker is placed for subsequent use.
Embodiment 2
The Structural Identification of TCPL polymer.
TCPL polymer identifies structure by hydrogen nuclear magnetic resonance and infrared spectrum.Fig. 1 (A), on the hydrogen spectrum spectrogram of hydrogen spectrum result display: TCPL, chemical displacement value 7.75-7.91ppm is the characteristic peak of TPP benzene ring hydrogen, the characteristic peak of chemical displacement value to be 2.27-2.55ppm the be hydrogen of methylene on PEI, the characteristic peak of chemical displacement value to be 5.84ppm the be hydrogen of methylene on LND, the characteristic peak of the hydrogen of methylene on chemical displacement value to be 3.71-3.81ppm be oxidation chitosan sugar chain, above-mentioned characteristic peak all has display, the synthesis of polymer TCPL is described successfully.
Fig. 1 (B), the results of FT-IR shows: TCPL is at 1404cm -1there is C=N stretching vibration peak, the formation of Schiff's base key is described.
Embodiment 3
PPX Macroscopic single crystal scheme is specific as follows: for folic acid FA.Take FA 0.1mmol, be dissolved in 0.1MNa 2cO 3in solution, then add 0.2mmol EDC, NHS respectively, stir, after activation 30min, be transferred to containing 0.1mmol H 2n-PEG-NH 20.1M Na 2cO 3in solution, stir, under room temperature, react 2h.Adopt the bag filter dialysis of molecular weight cut-off value 1000,0.01M Na 2cO 3solution is dialysis solution, dialyses without (ultraviolet detection) FA to outer liquid, then uses distill water dialysis 24h.PEG-FA is obtained after lyophilization.Take appropriate polyacrylic acid, be dissolved in sodium carbonate liquor, add EDC, NHS of polyacrylic acid carboxyl mole 4%, after 30min, add PEG-FA, react 2h under room temperature, dialysis postlyophilization obtains PPX (PPF), keeps in Dark Place.
Embodiment 4
The Structural Identification of PPX polymer.
PPX polymer identifies structure by hydrogen nuclear magnetic resonance.Fig. 2, hydrogen spectrum result display: in PPX hydrogen spectrum spectrogram, chemical shift 8.67ppm is the characteristic peak of hydrogen on folic acid pteridine, chemical shift 3.53ppm is the characteristic peak of hydrogen on PEG methylene, chemical shift 1.02-2.22ppm is the characteristic peak of methylene hydrogen on PAA, above-mentioned characteristic peak all has display, and the synthesis success of polymer P PX is described.
Embodiment 5
TCPL/siRNA/PPX self-assembled nanometer is total to the preparation of delivery system.
Preparation scheme is specific as follows: joined by siRNA solution in isopyknic TCPL solution under vortex, vortex 30s, room temperature leaves standstill 30min; Then get isopyknic PPX solution to add under vortex, vortex 30s, room temperature leaves standstill 30min, to obtain final product.
Embodiment 6
Self-assembled nanometer grain is investigated and the sign of nanoparticle siRNA binding ability
Nanoparticle is characterized by electrophoresis siRNA compression and protective capability.After TCPL/siRNA and TCPL/siRNA/PPX combines with different mass ratioes, add loading pigment, last volume is 10 μ L.Be added in 2% agarose gel, GelRed dyes, and with TAE buffer as electrolyte, runs 40min under 50V.
Fig. 3 (A)-(D), the pattern of complex passes through transmission electron microscope observing.Getting 1 TCPL/siRNA/PPX nanoparticle drips on copper mesh, with 1% uranyl acetate solution-dyed 10s.At electric Microscopic observation in the dry 10min of copper mesh.
The size of TCPL/siRNA/PPX nanoparticle and surface charge use Dynamic Light Scattering Determination.Particle diameter is about 120nm, and Zeta electric potential is-8mV about.
Embodiment 7
TCPL/siRNA/PPX nanoparticle HeLa cellular uptake and intracellular transport process
Fig. 4 (A), cellular uptake adopts flow cytometry analysis.HeLa cell is with every hole 1 × 10 5cells/well is inoculated in 24 orifice plates, cultivates 24h for 37 DEG C.Prepare FITC-TCPL/Cy3-siRNA/PPX nanoparticle according to nanoparticle preparation method, be adjusted to debita spissitudo by RPMI 1640 culture medium not containing folic acid.Discard the culture medium in every hole, add the sample solution of 1mL, continue to cultivate 4h.Discard sample solution afterwards, clean 3 times with cold PBS, for a moment, discard pancreatin, the PBS adding proper volume blows and beats into cell suspension to the trypsinization of every hole, after 300 order nylon net filters, carries out flow cytomery immediately.Medicine and siRNA can be delivered in same cell by result display nanoparticle.
Fig. 4 (B)-(C), intracellular transport process adopts confocal laser scanning microscope.HeLa cell is inoculated in laser co-focusing culture dish with every hole 5 × 104 cell, cultivates 24h for 37 DEG C.Containing RPMI 1640 culture medium of FITC-TCPL/siRNA/PPX nanoparticle, join in culture dish when cultivating 6h, discard sample solution, add 100nMMitoTracker Red and to dye 30min.After cell is dyed, discard dyeing liquor, clean 2 times with cold PBS, 4% paraformaldehyde solution fixes 20min, carries out confocal laser scanning microscope.Containing RPMI 1640 culture medium of FITC-TCPL/Cy3-siRNA/PPX nanoparticle, join 6h in culture dish, discard sample solution, clean 2 times with cold PBS, 4% paraformaldehyde solution fixes 20min, carries out confocal laser scanning microscope.Result is presented at Intracellular drug targeted delivery to mitochondrion, and siRNA discharges at kytoplasm.
Embodiment 8
Cytotoxicity
Adopt MTS method to analyze nanoparticle and carry siRNA and lonidamine to the impact of HeLa cell inhibitory effect.The good HeLa cell of growth conditions is with 1 × 10 4cells/well adds in 96 orifice plates, after incubated overnight, discard culture medium, add the culture medium containing TCP/siSCR/PPX nanoparticle, TCP/siBcl-2/PPX nanoparticle, lonidamine, CPL/siSCR/PPX nanoparticle, TCPL/siSCR/PPX nanoparticle, TCPL/siBcl-2/PPX nanoparticle and TCPL/siBcl-2/PP nanoparticle etc. respectively, the multiple hole of each sample at least 3, with normal cell as a control group, what only add culture medium is blank group.For making each processed group above-mentioned have comparability, fixed L ND concentration is 1.31 μ g/mL, and siRNA concentration is 0.5 μ g/mL.After cultivating 24h and 72h respectively, every hole adds the MTS solution of 20 μ L, and lucifuge 37 DEG C of jolting 4h, microplate reader 490nm detect absorbance (A).Fig. 5, cytotoxicity result shows, and lonidamine and siBcl-2 can work in coordination with the propagation suppressing HeLa cell.
Embodiment 9
Apoptosis
The research of nanoparticle cell death inducing is carried out according to the operation of AnnexinV-FITC cell apoptosis detection kit.HeLa cell is with every hole 3 × 10 5cell is inoculated in 6 orifice plates, after incubated overnight, every hole adds respectively containing TCP/siSCR/PPX nanoparticle, TCP/siBcl-2/PPX nanoparticle, lonidamine, CPL/siSCR/PPX nanoparticle, TCPL/siSCR/PPX nanoparticle, TCPL/siBcl-2/PPX nanoparticle and TCPL/siBcl-2/PP nanoparticle etc.For making each processed group above-mentioned have comparability, fixed L ND concentration is 1.31 μ g/mL, and siRNA concentration is 0.5 μ g/mL.After cultivating 48h, PBS cleans 2 times, and every hole adds not containing the pancreatin 200 μ L of EDTA, adds 1mL PBS by cell harvesting in EP pipe, 2000rpm, 4 DEG C of centrifugal 5min after sucking.Abandoning supernatant, it is resuspended that every hole adds 500 μ L Bindingbuffer.After every hole adds 5 μ L Annexin V-FITC mixings, then add 5 μ L Propidium Iodide, lucifuge reaction 10min under room temperature after mixing, and preparation singly contaminates pipe accordingly.Nylon membrane carries out flow cytomery after filtering immediately.Fig. 6, apoptosis result shows, and lonidamine and siBcl-2 can work in coordination with the apoptosis rate increasing HeLa cell.
Embodiment 10
The apoptosis-related protein of Western blotting detection line mitochondrial pathway
After nanoparticle solution-treated HeLa cell 48h, trypsinization is centrifugal, collecting cell.With appropriate cell pyrolysis liquid (containing 100 μ g/mL PMSF) re-suspended cell, place 1h on ice, abundant cell lysis.4 DEG C, the centrifugal 10min of 12000rpm, gets supernatant, adopts BCA method to detect protein content.Protein sample adds 6 × sample-loading buffer in proportion, and 100 DEG C of heating 5min make albuminous degeneration, get protein sample and join in right amount in loading hole.Wet method transferring film after electrophoresis, by film 1x Ponceaux dye liquor dye 5min after having turned, 1h closed by 5% skim milk.Add primary antibodie to hatch, primary antibodie (as Bcl-2, Bax, Caspase 3, Caspase 9) is respectively with 5% milk confining liquid dilution (1:500), and 4 DEG C are spent the night.Two anti-hatch: abandon primary antibodie, PBS-T washes film 3 times, at room temperature jolting on decolorization swinging table, each 10min.Film is hatched together with 1% milk IgG/HRP (two resist), 37 DEG C of jolting 2h.Add luminous nitrite ion after washing film with PBS-T on decolorization swinging table under room temperature, room temperature effect 1min, puts in gel imaging instrument and takes pictures.And with gel images processing system scan light density value.Fig. 7, result shows, and lonidamine and siBcl-2 can work in coordination with the apoptosis triggering mitochondria pathway.
Embodiment 11
Anti-tumor in vivo drug effect
H22 is inoculated in the right oxter of ICR male mice, treats that tumor grows to 100mm 3time, be divided into 7 groups at random, often organize 6, respectively tail vein injection administration.Experiment grouping is respectively: matched group (normal saline group), TCP/siSCR/PPX group, LND group (1.05mg/kg), LND group (40mg/kg), TCP/siBcl-2/PPX group, TCPL/siSCR/PPX group, TCPL/siBcl-2/PPX group.Dosage regimen: tumor-bearing mice oxter tumor grows to 100mm 3time start administration, count the 0th day with first time administration, respectively at administration in 0,2,4,6,8,10,12 day.Dosage: siRNA 0.2mg/kg, lonidamine 1.05mg/kg.After treatment terminates, dissect and take out tumor tissues, arrange according to every group, take pictures and weigh.Fig. 8, in body, tumor suppression result display lonidamine and siBcl-2 can work in coordination with the growth of Tumor suppression.
The above is only the preferred embodiment of the present invention; be noted that for those skilled in the art; under the premise without departing from the principles of the invention, can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

Claims (10)

1. send a nano-carrier altogether, form complex by polymeric prodrugs carrier TCPL and siRNA, and then formed with multi-functional polyanionic polymer PPX self assembly and send nano-carrier TCPL-siRNA-PPX altogether;
Wherein, the chemical structural formula of polymeric prodrugs carrier TCPL is as follows: wherein, and n, y, z are positive integer,
The chemical structural formula of multi-functional polyanionic polymer PPX is as follows:
Wherein, p, m 1, m 2for positive integer, X is target ligand, is selected from folic acid or lactobionic acid or RGD.
2. according to claim 1ly send nano-carrier altogether, preparation method comprises the following steps: joined by siRNA solution in isopyknic TCPL solution under vortex, vortex 30s, room temperature leaves standstill 30min; Then get isopyknic PPX solution with siRNA solution to add under vortex, vortex 30s, room temperature leaves standstill 30min, obtains and sends nano-carrier TCPL-siRNA-PPX altogether.
3. according to claim 1ly send nano-carrier altogether, it is characterized in that: described siRNA is Bcl-2siRNA, and/or; The phosphate buffer that described multi-functional polyanionic polymer PPX is dissolved in pH 7.4 forms PPX solution.
4. according to any one of claim 1-3, send nano-carrier altogether for the preparation of the purposes in Therapeutic cancer medicine.
5. a polymeric prodrugs carrier TCPL, is characterized in that: polymeric prodrugs carrier is that TPP is connected with amido link with PEI respectively with lonidamine, and PEI is connected with oxidation chitosan with Schiff's base key again, and its chemical structural formula is as follows:
Wherein, n, y, z are positive integer.
6. polymeric prodrugs carrier TCPL according to claim 5, is characterized in that: the synthetic method of described polymeric prodrugs carrier TCPL is as follows:
Wherein, the molecular weight of polymine PEI is 800-3500.
7. the polymeric prodrugs carrier TCPL according to claim 5 or 6, is characterized in that: the preparation method of described polymeric prodrugs carrier TCPL, specifically comprises the following steps:
1) synthesis of TPP-COOH: triphenylphosphine TPP and 6-bromocaproic acid feed intake with certain proportion mol ratio, are dissolved in anhydrous acetonitrile, and react 10-24h under nitrogen protection, recrystallization obtains TPP-COOH;
2) get appropriate TPP-COOH, anhydrous DMSO dissolves, and adds DCC and NHS, stirred at ambient temperature reaction 6-24h, centrifugal removing precipitation, and supernatant mixes with the anhydrous DMSO solution containing polymine PEI, and stirred at ambient temperature reaction 6-24h obtains reactant liquor;
3) appropriate amount of drug lonidamine LND is got, anhydrous DMSO dissolves, add DCC and NHS, stirred at ambient temperature reaction 6-24h, centrifugal removing precipitates to obtain supernatant, supernatant is mixed homogeneously with reactant liquor, continue stirring reaction 6-24h under room temperature, then the reactant liquor bag filter of molecular weight cut-off value 1000 is dialysed, and first dialyses with DMSO, dialyse respectively 1 time with the DMSO aqueous solution of different volumes ratio again, each 24h; Finally use distill water dialysis, dialysis solution obtains TPP-PEI-LND through lyophilization;
4) get appropriate TPP-PEI-LND, dissolve with DMSO; The acetate buffer of oxidation chitosan dropwise joins in the DMSO solution of TPP-PEI-LND, 4 DEG C of reaction 24-72h, then dialyse with the bag filter of molecular weight cut-off value 3500, distill water dialysis, filtering with microporous membrane, filtrate lyophilization obtains polymeric prodrugs carrier TCPL.
8. a multi-functional polyanionic polymer PPX, it is characterized in that polyacrylic acid PAA is connected with the Polyethylene Glycol PEG of two end band amino with target ligand X, its chemical structural formula is as follows:
Wherein, p, m 1, m 2for positive integer; X is target ligand, is selected from folic acid or lactobionic acid or RGD.
9. multi-functional polyanionic polymer PPX according to claim 8, synthetic route is as follows:
Wherein, polyacrylic acid PAA molecular weight is 2000-10000, and Polyethylene Glycol PEG molecular weight is 1000-6000.
10. multi-functional polyanionic polymer PPX according to claim 8 or claim 9, is characterized in that: the preparation method of described multi-functional polyanionic polymer PPX, specifically comprises the following steps:
1) target ligand X 0.1mmol, is dissolved in Na 2cO 3in solution, then add 0.1-2mmol EDC, NHS respectively, stir, after activation 10-60min, be transferred to containing 0.1mmol H 2n-PEG-NH 2na 2cO 3in solution, stir, under room temperature, react 1-12h; Adopt the bag filter dialysis of molecular weight cut-off value 1000, Na 2cO 3solution is dialysis solution, dialyses without X to outer liquid, then uses distill water dialysis 24h, obtain PEG-X after lyophilization;
2) take appropriate polyacrylic acid, be dissolved in sodium carbonate liquor, add EDC, NHS of polyacrylic acid carboxyl mole 4%, after 10-60min, add PEG-X, under room temperature, react 1-12h, dialysis postlyophilization obtains multi-functional polyanionic polymer PPX, keeps in Dark Place.
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