CN103566379A - Preparation and application of intracellular triggering reduction sensitive drug linked gene targeted co-carrier - Google Patents
Preparation and application of intracellular triggering reduction sensitive drug linked gene targeted co-carrier Download PDFInfo
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Abstract
The invention relates to an intracellular triggering reduction sensitive drug linked gene targeted co-carrier which is a gene and drug double-loaded system and comprises disulfide bond cross-linked polyethylene imine, a poly-lactide glycolide copolymer and a surface modified targeted micromolecule. According to the invention, the surface modified active targeted group of a carrier can target drugs to a tumor part and enrich the drugs; a disulfide bond cross-linked in a polymer can be broken in a cell under the condition of high reduction, and the structure of the carrier is disassembled so that the drugs are released. The intracellular triggering reduction sensitive drug linked gene targeted co-carrier disclosed by the invention can effectively load the gene and the hydrophobic micromolecular drug, ensure the stability of the gene and is degraded and releases the gene and the micromolecular drug under the condition of reduction. An in-vivo experiment shows that the intracellular triggering reduction sensitive drug linked gene targeted co-carrier disclosed by the invention has outstanding targeting on tumors.
Description
Technical field
The present invention relates to high molecular polymer genophore and gene therapy neck treatment field.Be specifically related to a kind of polymer for genophore and take that this polymer is basic engineering and the medication combined gene target of a kind of " born of the same parents' internal trigger " formula reduction responsive type that builds carrier altogether.The present invention relates to synthetic and sign, preparation method and the application thereof of carrier altogether of cross-linked type polyethylene imines.Carrier relates to complex, preparation method and the application that hydrophobic small molecules medicine, gene and nanoparticle form altogether.
Background technology
According to statistics, in Chinese malignant tumor, be the second largest killer who is only second to cardiovascular and cerebrovascular disease, and the first-selected excision of most patient, radiotherapy and chemotherapy.These therapies can obtain fast infantile tumour patient and effectively treatment, but to late period with local diffusion and transferrer, not life cycle of significant prolongation patient and improve patient's life quality, also find the toxic and side effects that chemotherapeutics is huge and the drug resistance problem in use producing simultaneously.In recent years, researcher finds that cancer is a kind of genopathy, is under the human body cell effect of environmental factors outside, and inherent multiple front oncogene is activated and the process of the multistage long term evolution of antioncogene inactivation.Therefore, gene therapy is significant for thorough radical cure tumor.
Gene therapy (gene therapy) refers to external source normal gene is imported into target cell by suitable carrier, to correct or to compensate because of genetic flaw and the disease that extremely causes, to reach therapeutic purposes.SiRNA (siRNA) is a class specificity and the double-stranded RNA that optionally suppresses expression of target gene, because of its specificity, high efficiency and as the great potential of gene therapy medicament, enjoys favor.At present, RNAi technology in the Etiologic Mechanism research of the various diseases such as nerve retrograde affection, diseases of cardiovascular and cerebrovascular systems, respiratory system disease, digestive system disease, hereditary, immune related diseases, diagnose aspect sharp treatment and all there is potential using value.The exploitation of the siRNA oligomerization medicine of synthetic is becoming a focus.
For specificity, suppress the expression of certain enzyme in tumor and do not disturb its siRNA expressing in normal cell, its carrier must possess tumor-targeting simultaneously.For example, cyclooxygenase (COX) is the crucial rate-limiting enzyme of arachidonic acid synthesis prostaglandin (PG).Structural type COX-1 expresses in histiocyte, and catalysis physiological prostaglandin synthesizes and the adjusting of participation body physiological function; The type COX-2 of inducing is easier to induction after hormone, inflammatory cytokine, somatomedin and tumor promotor cause cell-stimulating and produces.Large quantity research shows that the commitment that COX-2 forms in kinds of tumors is high expressed state, can developing by promotion tumors such as Angiogensis, acceleration tumor cell proliferation, inhibited apoptosis.Adopt RNAi technology to suppress the expression of the mRNA of coding COX-2, and then suppress the pirate recordings of COX-2 albumen, thereby can impel the apoptosis of tumor cell.Yet COX-2 all has expression and brings into play important physiological action in normal structure.Therefore, the siRNA that builds COX-2 must have an effect to tumor cell to targeting, and can not disturb the expression of COX-2 in normal cell.
Yet; although research is found gene therapy and people's normal gene or medicative exogenous gene can be imported and carried out effective expression in human body target cell by certain way; correct genetic flaw with treatment cancer; and be acknowledged as the effective means of current root curing cancers; in gene therapy process, easily there is Immunogenicity; therefore still need by with other traditional remedies, if surgical operation, radiotherapy, chemotherapy etc. are in conjunction with application, thus the advantage of performance Synergistic, Comprehensive Treatment.Because most anti-tumor small molecular medicine exists serious toxic and side effects, water solublity is low and without shortcomings such as targetings, its clinical practice has been subject to very big restriction.Recent researches discovery, macromolecular material can effectively address the above problem as anti-cancer medicament carrier, reaches good tumor killing effect, but it uses meeting generation drug resistance problem continuously.Therefore the targeting that, designs and build a kind of genomic medicine and micromolecule cancer therapy drug altogether carrier drug-supplying system has definite meaning.
The delivery technique of gene mainly contains following several at present: (1) direct transfection.Be injected directly in tissue or cell.In direct transfection process, gene is easy to be degraded, and needs heavy dose of application, may activated cell toxic reaction.(2) inactivation of viruses carrier transfection.Viral vector transfection efficiency is high, but has very serious safety problem, and genes of interest capacity is little, and preparation is complicated, and cost is high, can not the interior Reusability of body.(3) non-virus carrier transfection, comprises liposome transfection and polymer nanoparticle transfection.Non-virus carrier is owing to having low toxicity, low immunoreation, and exogenous origin gene integrator probability is low, without the restriction of gene Insert Fragment size, and use simple, easy to prepare, be convenient to preserve and the advantage such as check, be day by day subject to people and pay attention to.Under physiological condition, nucleotide sequence, with certain negative electricity, is easy to be adsorbed by electrostatic interaction by cation fragment, thereby can guarantee that gene carries the integrity in delivery process.Therefore cationic polymer has become the emphasis of furtheing investigate in non-virus carrier.In polymine (PEI) molecule, every two carbon atoms just should have a protonated nitrogen-atoms mutually, the pKa value of uncle's ammonia, parahelium and the tertiary amino group forming due to these nitrogen-atoms is different, makes PEI almost under any pH condition, have the ability of absorption proton.PEI " proton sponge " characteristic can absorb H in the sour environment of cell Inclusion
+, osmotic pressure in Inclusion is increased, cause that film is unstable even to break, thereby make the complex of being engulfed escape out, avoid Gene degradation, guarantee efficient transfection efficiency.
Reduced glutathion (GSH) is the abundantest biological micromolecule that contains sulfydryl of zooblast intensive amount, and reduced glutathion (GSH)/oxidized form of glutathione (GSSG) is topmost redox couple in body.Inside and outside cell, in different environment there is significant difference in the content of GSH/GSSG.Due to the effect of the sharp glutathion reductase of DPNH I (NADPH), in cell, GSH concentration is 100-1000 times of extracellular environment, thereby makes intracellular environment keep stronger reproducibility.In addition, research discovery, in tumor tissues, GSH concentration ratio normal structure is at least high 4 times, therefore, than normal group, is woven with higher reproducibility.In the present invention, reducing environment responsive type nanoparticle transmission system is the polymine based on disulfide bond crosslinking, the switch of stimulate disulfide bond as reducing environment-response, makes its stable existence under extracellular environment, and under reducing environment, ruptures in tumor cell.
Biodegradable material polylactic acid/hydroxy acetate multipolymer (PLGA) is that lactic acid and hydroxyacetic acid combined polymerization form, there is good stability and biocompatibility, be easy to by cytophagy, micromolecule hydrophobic drug is had to good envelop rate and drug loading, can effectively solve the bad problem of water solublity that antitumor drug exists, can not only increase the dissolubility of insoluble drug simultaneously, and can increase stability and the dissolution rate of medicine, can change even to a certain extent kinetics and the pharmacological properties of medicine.
Targeting drug delivery system is the Novel Drug Delivery Systems that a class can make medicine concentrate to be positioned pathological tissues, organ, cell or cell inner structure, is the first-selected drug-supplying system of antitumor drug.Its targeting mechanism mainly contains passive target, active targeting and physical chemistry targeting.Passive target is mainly the EPR effect of utilizing tumor locus, by controlling preparation size, accumulates in tumor locus; Physical chemistry targeting refers to controlling magnetic field, heat, light, pH etc., thereby makes drug-supplying system discharge medicine at tumor locus; Initiatively targeting is the targeted system designing according to tumor cell surface receptor.For film polysaccharide or the memebrane protein of tumor cell surface overexpression, with polysaccharide, drug-supplying system is modified, can obviously strengthen the affinity to tumor cell.Hyaluronic acid (HA) is a kind of native protein polysaccharide extensively distributing in the organ of extracellular matrix, connective tissue and higher mammal.CD44 is a class transmembrane glycoprotein, is the most important HA receptor of cell surface.Research at present shows kinds of tumor cells apparent height expression CD44 molecule.The feature of utilizing HA water solublity and tumor cell targeting, has become one of antitumor drug study hotspot using it as pharmaceutical carrier.
Summary of the invention
One of object of the present invention is to provide a kind of reduction sensitive polymer material, it is characterized in that low molecular weight polyethylene imines obtains by disulfide bond crosslinking; This polymer and polylactide glycolide copolymer form nanoparticle, simultaneously at its pan coating hyaluronic acid.This delivery system simultaneously initiatively targeted delivery gene and micromolecule hydrophobic drug to tumor locus, in tumor cell, disulfide bonds discharges medicine and gene, thereby reduction toxicity, improves medicine and gene concentration in tumor, reaches good therapeutic effect.
Two of object of the present invention is to provide the medication combined gene target of a kind of " born of the same parents' internal trigger " formula reduction responsive type preparation method of carrier altogether.
Three of object of the present invention is to provide the application of carrier in antitumor field altogether of the medication combined gene target of this " born of the same parents' internal trigger " formula reduction responsive type.
The invention provides following technical scheme:
(1) polymine and disulfide bond crosslinking reagent reacting, obtain disulfide bond crosslinking type low molecular weight polyethylene imines (PEIss).Under this reaction needed nitrogen protection, lucifuge condition, carry out.Described polymine also comprises its derivant.
(2) disulfide bond crosslinking type low molecular weight polyethylene imines and polylactide glycolide copolymer are prepared nanoparticle by emulsion-solvent evaporation method.
(3) hyaluronic acid covers nanoparticle surface by covalent bond or non-covalent bond.Described hyaluronic acid also comprises its derivant.
Cross-linking agent for the synthesis of disulfide bond crosslinking type low molecular weight polyethylene imines in the present invention can be dithiodipropionic acid succimide ester, dimethoxy-dithio propyl group inferior amine salt hydrochlorate, N, N '-cystamine bisacrylamide, preferred N, N '-cystamine bisacrylamide; Polymine molecular weight is 1800Da～750,000Da, preferably 1800Da; Reaction dissolvent can be DMSO, DMF, methanol, water, particular methanol-water (80%); The molar ratio scope of cross-linking agent and polymine is 1:4～1:1, preferably 1:2; Range of reaction temperature is room temperature～60 ℃; Response time is 2-10 days, preferably 3 days; Reaction requires to carry out under lucifuge, nitrogen protection condition.Synthetic reaction purification, both can adopt bag filter dialysis purification, also can adopt centrifugal concentrating pipe purification, and preferably centrifugal concentrating pipe, to save time, is convenient to lyophilization.
The present invention adopts emulsion-solvent evaporation method to prepare nanoparticle, and the organic facies solvent of employing can be the mixed solvent of dichloromethane, ethyl acetate, acetic acid, acetone and above-mentioned several solvents, preferably dichloromethane; The surfactant adopting can be poloxamer, polyvinyl alcohol, Tween 80 etc., preferred PLURONICS F87, and concentration is 1% to 10%, preferably 5%; Emulsification method can adopt ultrasonic method or high speed shear method, preferred ultrasonic method, and emulsification times is 5min to 30min, preferably 15min; Polylactide glycolide copolymer molecular weight is 5000Da to 40000Da, and block ratio is 50:50,75:25, preferred molecular weight 10, and 000Da, block compares 50:50; Polylactide glycolide copolymer and disulfide bond crosslinking type low molecular weight polyethylene imines mass ratio are 1:1 to 20:1, preferably 10:1.
The present invention adopts hyaluronic acid to be covered in nanoparticle surface by covalent bond or non-covalent bond.Covalent bond modifies preferred 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate and N-hydroxy-succinamide carries out acylation reaction by amino on the carboxyl on hyaluronic acid sugar ring and disulfide bond crosslinking type low molecular weight polyethylene imines.Non-covalent bond is modified and is preferably passed through electrostatic interaction, and the disulfide bond crosslinking type low molecular weight polyethylene imines of electronegative hyaluronic acid and positively charged is hatched to certain hour.
The present invention take various hydrophobic micromolecule cancer therapy drug (as paclitaxel, docetaxel, amycin etc.) wherein a kind of be model, prepare according to the method described above drug-carrying nanometer particle, drug loading is 50%-100%, 24-120h can discharge completely.
The invention provides the medication combined gene target carrier of " born of the same parents' internal trigger " formula reduction responsive type applies in gastric cancer, hepatocarcinoma, pulmonary carcinoma, breast carcinoma, carcinoma of gallbladder, straight adenocarcinoma of colon, adenocarcinoma of esophagus, cervical cancer, cerebral astrocytoma.
Accompanying drawing explanation
Fig. 1 is that in the present invention, disulfide bond crosslinking type low molecular weight polyethylene imines utilizes AVANCE500MHZ nmr determination
1hNMR scintigram.
Fig. 2 is form and the grain-size graph that in the present invention, nanoparticle utilizes atomic force microscope to measure.
Fig. 3 is the binding ability electrophoretogram that in the present invention, nanoparticle and siRNA utilize agarose gel electrophoresis to measure, left figure is that electrophoretic band brightness integration is converted into the combination percent curve that optical density value is drawn, right figure is electrophoresis image, by electrophoresis tank path from left to right sequentially, and N/P=0,0.25.0.5,1,2,5,10,20.
Fig. 4 is that in the present invention, nanoparticle utilizes agarose gel electrophoresis to investigate its nuclease-resistant degradation capability electrophoretogram after being combined with siRNA, and left figure is converted into the degraded percent curve that optical density value is drawn, right figure a by electrophoretic band brightness, b, c application of sample is respectively naked siRNA, PLGA-PEIss/siRNA complex and PLGA-PEI/siRNA complex, electrophoresis tank path application of sample is from left to right respectively complex and Rnase hatches 0,5,15,30,45, product after 60,120,240min.
Fig. 5 reduces in the present invention after responsive type nanoparticle is combined with siRNA to utilize agarose gel electrophoresis to investigate its reduction-sensitive, upper figure application of sample is PEI-PLGA/siRNA complex, figure below application of sample is PEIss-PLGA/siRNA complex, electrophoresis tank path application of sample is from left to right respectively U/ug (heparin sodium/siRNA)=0,0.015,0.03,0.0375,0.075,0.15,0.3.
Fig. 6 carries the reduction sensitive nanoparticles release in vitro curve of docetaxel in the present invention.
Fig. 7 is that reduction sensitive nanoparticles hyaluronic acid decorated in the present invention distributes in soma.
The specific embodiment
Below in conjunction with embodiment, with disulfide bond crosslinking reagent: N, N '-bis-(third rare acyl) cystamine is example, but is not limited to this, and invention is elaborated:
Synthetic and the sign of disulfide bond crosslinking type low molecular weight polyethylene imines
(1) synthesis condition
Take polymine (Mw=1800) lg (0.56mmol), be placed in three neck round-bottomed flasks, add methanol-water (80%) 9ml to make to dissolve.Take N, N '-bis-(third rare acyl) cystamine 0.07g (0.28mmol), adds methanol-water (80%) 1ml to make to dissolve, and slowly drops in three-neck flask.Stir 55 ℃, nitrogen protection, lucifuge reaction 3 days.Dialysis purification, lyophilization obtains disulfide bond crosslinking type low molecular weight polyethylene imines.
(2) structural characterization
Get the dry sterling of disulfide bond crosslinking type low molecular weight polyethylene imines appropriate, be dissolved in D
2in O, by nuclear magnetic resonance, NMR (
1h-NMR) it is carried out to result confirmation.Polymine
1in H-NMR collection of illustrative plates, characteristic peak concentrates on 2.5～2.9 places; And the low molecular weight polyethylene imines of disulfide bond crosslinking
1in H-NMR collection of illustrative plates, in δ=3.47,3.08,2.38 peaks that occur belong to respectively the methylene peak of CBA and the methylene that the two keys of acrylamide generate after Michael additive reaction.The results are shown in Figure 1.
The preparation of nanoparticle and quality evaluation
(1) preparation of nanoparticle
Preparation 20mg/ml disulfide bond crosslinking type low molecular weight polyethylene imines acetone storing solution: take disulfide bond crosslinking type low molecular weight polyethylene imines 0.2g, add 0.05g Tween 80, be dissolved in 10ml acetone lucifuge, ultrasonic dissolution.
Organic phase solution preparation: (10,000Da50:50) 0.2g, adds 0.1g Tween 80, is dissolved in 2ml dichloromethane ultrasonic dissolution to take polylactide glycolide copolymer.Add disulfide bond crosslinking type low molecular weight polyethylene imines acetone storing solution 1ml, add acetone to 10ml, mix.
Aqueous phase solution preparation: take 0.5g PLURONICS F87, be dissolved in 10ml water.
Organic facies is joined in water, use Ultrasonic cell smash by the ultrasonic 15min of mixed solution.45 ℃ revolve steaming 5min, remove organic solvent.0.45 μ m filter membrane filters, and obtains blank nanoparticle.
In organic phase solution, add a certain amount of docetaxel acetone soln, obtain drug-carrying nanometer particle.
(2) quality evaluation of nanoparticle
AFM Analysis, is diluted to suitable concentration by blank nanoparticle, drips on clean coverslip, and room temperature hold over night, air-dry.Be placed under atomic force microscope, microexamination nanoparticle form and particle diameter after regulating, the results are shown in Figure 2.
Embodiment 3
Hyaluronic modification and external inner evaluation
(1) hyaluronic acid decorated
Non-covalent combination: get drug-carrying nanometer particle appropriate, add hyaluronic acid solution, blending incubation 15min, obtains hyaluronic acid decorated nanoparticle.
Covalent bond: appropriate hyaluronic acid solution and drug-carrying nanometer particle are mixed, add the hydrochloric acid of 1.0mol/L, make the pH value to 6.5 of reactant.Separately get appropriate 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate and N-hydroxy-succinamide, be dissolved in dimethyl sulfoxide-water (50%), slowly add in reactant solution, stirring at normal temperature is mixed after 24h again, with the sodium hydroxide of 1.0mol/L, regulates pH.After dialysis, lyophilization obtains hyaluronic acid decorated nanoparticle.
The nanoparticle of getting after hyaluronic acid decorated is appropriate, mixes by a certain percentage with siRNA solution, hatches 15min.Obtain two drug-carrying nanometer particles.
(2) Evaluation in Vivo and in Vitro
Agarose gel electrophoresis is analyzed, and gets hyaluronic acid decorated blank nanoparticle, and pressing N/P ratio (N:P) with siRNA is 0:1,0.25:1,0.5:1,1:1,2:1,5:1,10:1,20:1 mixes, and after hatching, both electrostatical binding abilities of agarose electrophoretic analysis, the results are shown in Figure 3.
Agarose gel electrophoresis is analyzed, and by N:P, than being 10:1 preparation PLGA-PEI/siRNA and PLGA-PEI-ss/siRNA nanoparticle complex solution, cumulative volume is 10uL, and every part of solution is containing siRNA0.4ug.RNase solution (40ug/ml) is added to complex, and under 37 ℃ of conditions, hatch respectively 0,5,15,30,60,120,240min.When reaction finishes, deposit in immediately-20 ℃ of refrigerators.Treat that the sampling of All Time point is complete, add heparin solution (10%) 1ul, make U/ug (heparin sodium/siRNA)=0.0375, siRNA dissociates out from complex.Get above-mentioned mixed liquor and in 2% agarose gel, carry out electrophoresis.Voltage 85V, electrophoresis time is 30min, under uviol lamp, observes electrophoresis result.Do the negative control of naked siRNA simultaneously.Utilize gel imaging software that electrophoretic band brightness is converted into optical density value and draw degradation kinetics curve, calculate the degradation rate constant of complex in nuclease.The results are shown in Figure 4.
Agarose gel electrophoresis is analyzed, fresh preparation DNA loaded nanoparticle, and adding reduced glutathion (GSH) to make final concentration is 20%.Nanoparticle is disulfide bonds under the effect of reduced glutathion (GSH), and carrier framework is destroyed, thereby discharges siRNA.Hatch after 30min for 37 ℃, draw 1ul according to different U/ μ g (heparin sodium/siRNA)=0,0.015,0.03,0.0375,0.075,0.15,0.3 adds heparin sodium aqua, and making final volume is 10ul.Agarose gel electrophoresis is investigated reduction-sensitive, the results are shown in Figure 5.
Get a year docetaxel nanometer grain and put in right amount bag filter, respectively with pH7.4, the phosphate buffer of pH5.8 is release medium (containing 0.5%Tween80,5% calf serum), is placed in 37 ℃ of constant temperature oscillation casees.Respectively at different time points 0.5h, 1h, 2h, 4h, 8h, 12h, 18h, 24h, 72h, gets 1ml sample stored refrigerated, adds 1ml acetonitrile simultaneously again, and HPLC method is measured free drug content, and calculates its accumulative total release rate, the results are shown in Figure 6.
The nanoparticle of getting hyaluronic acid decorated front and back is appropriate, carries respectively with fluorescent dye Dir.Get 15 of H22 tumor-bearing mices, give respectively 0.2ml normal saline, carry the PLGA-PEI nanoparticle of Dir, carry the PLGA-PEI-HA nanoparticle of Dir and the PLGA-PEIss nanoparticle that carries Dir, carry after the PLGA-PEI-HA nanoparticle 8h of Dir, dissect Organs of Mice and in living imaging instrument, carry out fluorescence intensity observation and calculate average fluorescent strength, the results are shown in Figure 7.
Claims (14)
1. the medication combined gene target carrier of " born of the same parents' internal trigger " formula reduction responsive type, is characterized in that, described carrier is the nanoparticle transmission system of chemicals and gene administering drug combinations; Polymer can pass through disulfide bond crosslinking, and modifies by active targeting group is coated on its surface.
According to claim 1 in carrier the introducing of disulfide bond including but not limited to dithiodipropionic acid succimide ester, dimethoxy-dithio propyl group inferior amine salt hydrochlorate, N, N '-cystamine bisacrylamide.
According to claim 1 in carrier initiatively the modification of targeting group can be polysaccharide as hyaluronic acid, mannose etc., or polypeptide protein class is as RGD sequence, transferrins etc.Wherein the modification of targeting group can be covalent bonding or non-covalent bond bonding.
4. carrier as described in claim 1, it is characterized in that polymine (polyethylenimines, PEI) or the polymine of disulfide bond crosslinking (PEIss) and polylactide glycolide copolymer (poly (lactic-co-glycolic acid), PLGA) mass ratio is 1:1-1:60, and wherein initiatively the molecular weight of targeting group is 3000 dalton～1,500,000 dalton; Polymine (or polymine of disulfide bond crosslinking) molecular weight ranges is 600 dalton～70,000 dalton; Polylactide glycolide copolymer molecular weight ranges is 5000 dalton～10,000 dalton, wherein lactide: the ratio of Acetic acid, hydroxy-, bimol. cyclic ester is 25:75～75:25.
5. desired disulfide bond crosslinking type low molecular weight polyethylene imines preparation method in right 1, comprises the following steps:
(1) polymine and cross-linking agent are dissolved in respectively in suitable reaction dissolvent to stirring reaction certain hour under uniform temperature, lucifuge, nitrogen protection condition according to certain mol proportion example.
(2) after reaction finishes, purification that reaction solution is dialysed in proper dialysis liquid, lyophilization obtains the polyethylene imine copolymer of disulfide bond crosslinking.
6. preparation method as described in claim 5, its reaction dissolvent can be atent solvent, as DMSO, DMF, methanol, water, particular methanol-water (80%); Reaction requires to carry out under lucifuge, nitrogen protection condition.The molar ratio scope of cross-linking agent and polymine is 1:4～1:1, and range of reaction temperature is room temperature～60 ℃, and the response time is 24～120h, and dialysis solution is water or inorganic salts, the dry freeze-drying that adopts.
7. the preparation method of desired nanoparticle carrier in right 1, comprises the following steps:
(1) polymine (PEIss) of polylactide glycolide copolymer and hydrophobic small molecules medicine, polymine (PEI) or disulfide bond crosslinking is dissolved in respectively in suitable organic solvent or aqueous solvent according to certain mass ratio, and adds a certain proportion of tween 80.To after above-mentioned solution mix homogeneously, slowly join in the aqueous solution of finite concentration surfactant.Under ice-water bath condition, use Ultrasonic cell smash to be interrupted after ultrasonic several minutes, on Rotary Evaporators, decompression evaporates organic solvent, obtains carrying PLGA-PEI nanoparticle or the PLGA-PEIss nanoparticle of small-molecule drug.With different N: P, nanoparticle is mixed homogeneously with gene, after incubated at room certain hour, by electrostatic self-assembled, form the two medicine carrying PLGA-PEI of gene-small-molecule drug or PLGA-PEIss complex solution.
(2) active targeting small-molecule substance is dissolved in to the solution that is mixed with 10% (W/V) in deionized water; under stirring condition, this solution is mixed with two drug-carrying nanometer particles; room temperature lucifuge stirs 24h， winner moving-target to PLGA-PEI nanoparticle or the PLGA-PEIss nanoparticle of base group modification.
8. preparation method according to claim 7, the polymine quality of polylactide glycolide copolymer and polymine or disulfide bond crosslinking is 5:1-60:1 than scope; Polylactide glycolide copolymer concentration range is 0.1mg/ml-10mg/ml; Tween 80 concentration range is 0.01%-1%; The kind of surfactant is: PLURONICS F87, and polyvinyl alcohol, concentration range is 0.1%-5%.
9. carrier altogether according to claim 7 is characterized in that particle diameter is less than 2 μ m.
10. method according to claim 7, the solvent that it is characterized in that dissolving common carrier is phosphate buffer, HEBS (4-hydroxyethyl piperazine ethanesulfonic acid) buffer, sodium chloride solution or glucose solution; The concentration range of PLGA-PEI nanoparticle or PLGA-PEIss nanoparticle and gene is 0.01mg/ml-10mg/ml, and N:P is 1:1-1:100 than scope; Initiatively the molar ratio range of the polymine of targeting small-molecule substance and polymine or disulfide bond crosslinking is 1:10-10:1; The drug loading of hydrophobic small molecules medicine is 1%-10%.
11. comprise the antitumor drug such as paclitaxel, docetaxel, amycin and hormone medicine, Chinese medicine monomer etc. according to the hydrophobic small molecules medicine described in the requirement of right 1.
12. according to the gene described in the requirement of right 1 can be can be recombinant expressed in eukaryotic cell containing reporter gene, antioncogene, cytokine gene plasmid DNA, oligonucleotide or siRNA.
13. application of altogether carrier according to claim 9 in transfectional cell, purposes is can successful transfection human body and endotheliocyte, epithelial cell and the kinds of tumor cells etc. of animal origin.
14. application of carrier altogether according to claim 9 in the corresponding disease medicine of preparation treatment, the route of administration of this common carrier comprises: injection, oral and mucosal drug delivery, be applied to the isogenic chemicals Polymorphism of tumor Synergistic treatment.
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| CN104262638A (en) * | 2014-09-02 | 2015-01-07 | 国家纳米科学中心 | Hyaluronic acid-cystamine-polylactic acid-glycollic acid graft polymer and preparation method thereof |
| CN104262638B (en) * | 2014-09-02 | 2017-01-25 | 国家纳米科学中心 | Hyaluronic acid-cystamine-polylactic acid-glycollic acid graft polymer and preparation method thereof |
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| CN110343255A (en) * | 2019-08-21 | 2019-10-18 | 温州医科大学 | Polymer support and preparation method thereof, anti-tumor nano particle |
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| CN111481679A (en) * | 2020-04-21 | 2020-08-04 | 河南大学 | siRNA nanocapsule and preparation method and application thereof |
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| CN113633785A (en) * | 2021-08-27 | 2021-11-12 | 中国药科大学 | Preparation method and application of intelligent responsive shell-core polyelectrolyte nanogel |
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| CN113995851A (en) * | 2021-10-21 | 2022-02-01 | 中国中医科学院中药研究所 | Preparation method of bionic siRNA nano-composite with anticancer activity |
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