CN105147615A - Tumor cell and tumor vessel double-target nanoparticle, building method and application - Google Patents
Tumor cell and tumor vessel double-target nanoparticle, building method and application Download PDFInfo
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Abstract
The invention belongs to the field of tumor targeted medication, and relates to a tumor cell and tumor vessel double-target nanoparticle, a building method and application. The invention discloses preparation and application of a DNA (Deoxyribonucleic Acid) tumor targeted cationic polymer nanoparticle (mPEG-PLGA-PLL-LA-VEGFab) of mPEG-PLGA-PLL modified by LA and VEGFaB. The mPEG-PLGA-PLL-LA-VEGFab nanoparticle is simultaneously modified by two targeting molecules, has the specificity targeted tumor tissue cell and tumor vessel characteristics, can be used for preparing targeted delivery anti-tumor medicine, and is particularly applicable to miRNA loading for preparing medicine for treating liver cancer. A preparing method is simple and convenient, and is suitable for mass production.
Description
Technical field
The invention belongs to Tumor Targeting Drug Delivery System technical field.Relate to a kind of tumor cell and the two targeted nano granule of tumor vessel, construction method and application.Be that methyl ether PEG-PLGA-polylysine (mPEG-PLGA-PLL) that lactobionic acid (LA) and VEGF antibody (VEGFab) are modified carries genetic tumour targeted nano granule (mPEG-PLGA-PLL-LA-VEGFab), preparation method and the application in field of medicaments thereof furtherly, be particularly useful for cancer therapy drug and the preparation of preparing injection for intravenous.
Background technology
Treatment with chemotherapy Drug therapy malignant tumor obtains certain success in many cases clinically, but, also there are some serious problems simultaneously.A main problem is that clinical antitumor drug generally lacks targeting, easily causes the generation of serious dose dependent toxic and side effects, significantly limit the antitumous effect of medicine.Therefore, how safely, deliver drugs into target cell and play a role efficiently is one of the most key factor obtaining desirable antineoplaston effect.
Carrier at present for drug delivery system is mainly divided into viral vector and non-virus carrier.Limit it greatly further apply because viral vector exists the problem such as toxic and side effects and potential immunogenicity, oncogenicity, thus there is non-immunogenicity, be easy to the non-virus carrier of the advantages such as preparation, good stability and be subject to domestic and international researcher and more pay close attention to.Picture non-virus carrier, as the nano-delivery systems such as liposome, nanoparticle, cationic polymer, micelle all achieve achievement in research in various degree in gene delivery.Nano-delivery system can the enhancing gene targeting of sending, be coupled specific targeted molecular on nano-carrier surface simultaneously, as ligands specific, monoclonal antibody etc., be combined with cell surface specific receptor by targeted molecular, the specific active targeting that can realize gene is sent, and improves therapeutic efficiency.It can thus be appreciated that the nonviral gene delivery carrier of good delivery efficiency and targeting, has very important effect in the gene therapy of tumor.Although then non-virus carrier has obtained larger progress, the problem of its specific targeting and efficient load effciency has not still been solved thoroughly.For this reason, the present inventor build on early-stage Study basis there is tumor high specific and high load gene efficiency mPEG-PLGA-PLL-La-VEGFab nanometer delivery vector (
peifengLiuetal.Intracellulartraffickingandcellularuptakemechanismo fmPEG-PLGA-PLLandmPEG-PLGA-PLL-Galnanoparticlesfortarget eddeliverytohepatomas.Biomaterials2014,35 (2): 760-770.), wherein the targeting design of mPEG-PLGA-PLL-La-VEGFab nano-delivery system will be realized by tumor tissue cell, tumor vascular multiple targeting, and this will improve the efficiency of gene delivery and oncotherapy effectively.
(1) macrocyclic tissue target to
The targeting of tumor tissues has the nanoparticle of long circulating characteristic in body by use and realizes, its passive target function mainly utilizing nanoparticle to have and produce enhancing infiltration be detained (EPR) effect, concrete grammar is that the nano-delivery system using mPEG to modify can escape engulfing of reticuloendothelial system (RES) in vivo effectively, increases delivery system circulation time in vivo and stability.
Degradable polymer PLGA has good biocompatibility, degradability and slow-releasing, and the inside and outside being applicable to gene is sent.Cationic polymer PLL PLL good biocompatibility, its flexible structure (polymer exists as configurations such as linear, star, random netted, block, grafting), stable, molecular weight are easily adjusted; And can easily by introducing side chain or the performance adjusting and improve carrier by the modification of specific target tropism group.
MPEG, PLGA and PLL are combined the advantage that can play three, the PLL that water solublity is stronger modifies fat-soluble PLGA, can improve hydrophilic and the crystallinity of PLGA, regulate its degradation speed, the block copolymer with parents characteristic formed then more easily forms nano-particle.MPEG can form one deck hydration shell on nanoparticle surface, hinders the absorption of plasma fraction and micellar surface, thus reduces reticuloendothelial system to the picked-up of nanoparticle, makes the time lengthening of nanoparticle in body circulation, increases the targeting to tumor.
(2) tumor cell and tumor endothelial blood-vessels target
In order to strengthen the high efficiency of nanoparticle targeting, we select tumor cell and the common targeting of tumor vessel.Utilize the specific receptor ligand combination principle of tumor cell, therefore select to tumor cell, to there is the lactobionic acid (La) of good target function and carry out decorated nanometer grain to the VEGF antibody (VEGFab) that tumor vessel has a good targeting.
Asialo-glycoprotein receptor (ASGP-R) is the specific receptor at hepatoma cell membrane surface high level expression, and normal cell and most normal organ system are then without expressing, and therefore ASGP-R is a desirable target.Theoretical according to receptor, the aglucon analog containing La basic sequence effectively can be combined with ASGP-R (combination of itself and aglucon carries out covalent bond by La basic sequence with ASGP-R to be combined).VEGF (VEGF) be a kind of can the factor of induced strong angiogenic growth, play an important role in tumor-blood-vessel growth process.Tumor cell can secretion of VEGF in hypoxia (hypoxia) situation.VEGF can promote endothelial cell proliferation, promotes cell migration, and inhibited apoptosis.Always the angiogenic growth situation of testing in the expression and hepatic carcinoma confirming VEGF is proportionate more, and is proportionate with shifting risk of tumor.And VEGFab is by effectively identifying and testing the object of active targeting in conjunction with the VEGF of tumor vascular endothelial cell.
It can thus be appreciated that selected mPEG-PLGA-PLL-La-VEGFab nanoparticle has powerful function.This project is also sent the anti-liver cancer treatment of miRNA to mPEG-PLGA-PLL-La-VEGFab nanoparticle simultaneously and is studied accordingly, for the clinical practice of this nanoparticle lays the foundation.
Summary of the invention
The object of this invention is to provide a kind of with methyl ether PEG-PLGA-polylysine-lactobionic acid-VEGF antibody mPEG-PLGA-PLL-La-VEGFab nanometer drug delivery sys tems, mPEG has long circulating effect, PLGA energy useful load antitumor drug, biodegradable have slow controlled release effect, positively charged can the mediation of PLL is combined with the gene of negative charge, LA energy efficient targeting hepatoma carcinoma cell, VEGFab energy efficient targeting tumor vessel, this makes the mPEG-PLGA-PLL-La-VEGFab nanometer drug delivery sys tems of structure have the multifunctional targeted function of tumour-specific.This carrier material also has functions such as transporting miRNA, siRNA, Chinese medicine monomer, ginsenoside and vascellum esoderma inhibin.
Technical problem to be solved by this invention is to select suitable carrier, can jointly be targeted to tumor tissue cell and tumor vessel, and effectively gene, medicine and Chinese medicine monomer is delivered to target spot.
1. mPEG-PLGA-PLL-LA-VEGFab of the present invention is the carrier material of a series of different molecular weight, different monomers ratio, and polymer molecular weight is 2.0 × 10
3-10.0 × 10
6; Wherein the weight ratio of mPEG/PLGA is 1-50:50-100, in PLGA, the weight ratio of LA/GA is 1-100:1-100, the weight ratio of PLGA/PLL is 50-100:1-50, mPEG-PLGA-PLL, the weight ratio of LA and VEGFab is 1:0.0001-0.001:0.0001-0.001.
In the two targeted nano granule mPEG-PLGA-PLL-LA-VEGFab of targets neoplastic cells of the present invention and tumor vessel, LA content is 0.01-10% weight, and VEGFab content is 0.01-10% weight, and all the other are the weight of mPEG-PLGA-PLL.The weight ratio of mPEG-PLGA-PLL, LA and VEGFab is 1:0.0001-0.001:0.0001-0.001.
In the two targeted nano granule of tumor cell of the present invention and tumor vessel, the method for attachment of mPEG-PLGA-PLL cationic polymer and LA and VEGFab forms covalent bond by the functional group reactions between cationic polymer and the polypeptide of LA and VEGFab to combine.
2. the construction method of two target cationic polymer nanoparticles of methyl ether PEG-PLGA-polylysine-lactobionic acid-VEGF antibody mPEG-PLGA-PLL-LA-VEGFab of the present invention: get mPEG-PLGA-PLL and be dissolved in organic solvent, add LA, Chinese named EDC and NHS stirring reaction 2-24h subsequently.Reaction terminate after by solution as the 3-96h that dialyses in bag filter, lyophilizing subsequently preserve.The two target cationic polymer nanoparticle of preparation organic solvent used is organic solvent is dimethyl sulfoxine, dichloromethane, chloroform and methanol; The weight ratio of mPEG-PLGA-PLL, LA, VEGFab, EDC and NHS is 1:0.0001 ~ 0.001:0.0001 ~ 0.001:0.001 ~ 0.01:0.001 ~ 0.01; The molecular cut off of bag filter is 600-2000.
MPEG-PLGA-PLL-LA-VEGFab nano-carrier of the present invention can prepare nanoparticle under the effects such as ultrasonic emulsification, dialysis, co-precipitation, particle diameter is controlled at 10 ~ below 1000nm, between prioritizing selection 10-300nm, smooth surface, good evenness, regular particles is without adhesion, redispersibility is good, drug loading and envelop rate high, can be used for the slow release nano-particle preparing vein or intramuscular injection or oral administration, as cancerous cell target administration.The nanoparticle of preparation can be dispersed in solid, semisolid or solution.Preferably make the pharmaceutical dosage forms of drug administration by injection, especially injection for intravenous is used.
MPEG-PLGA-PLL-LA-VEGFab administration nano-drug administration system of the present invention, can be prepared by following preparation method:
Multi-emulsion method: 4mgmPEG-PLGA-PLL-La-VEGFab carrier is dissolved in the organic solvent of 1.5mL ethyl acetate, dichloromethane or dichloromethane and acetone mixing, add pharmaceutical aqueous solution, ultrasonic or high pressure is newborn evenly obtains colostrum, add the Pluronic F68 water dispersion medium that 4mL concentration is 1% again, after emulsifying obtains emulsion, stir or rotary evaporation removing organic facies, obtain the mPEG-PLGA-PLL-La-VEGFab nanoparticle of load medicine.
Coprecipitation: be dissolved in by 4mgmPEG-PLGA-PLL-La-VEGFab carrier in 2mL acetone, be added drop-wise in the water dispersion medium of drug containing under stirring condition, stirs and waves most organic solvent, obtain the mPEG-PLGA-PLL-La-VEGFab nanoparticle of load medicine.
Emulsion solvent diffusion method: 4mgmPEG-PLGA-PLL-La-VEGFab carrier is dissolved in 2mL acetone/dichloromethane mixed solvent, join drug containing water-soluble in ultrasonic, then ultrasonic rear solution being joined again concentration is in the Pluronic F68 water dispersion medium of 1%, the newborn even emulsifying of ultrasonic or high pressure, emulsion at room temperature waves most organic solvent, obtains the mPEG-PLGA-PLL-La-VEGFab nanoparticle of load medicine.
Or getting mPEG-PLGA-PLL-La-VEGFab is dissolved in the mixed solution of dichloromethane or dichloromethane and acetone, add miRNA, ultrasonic emulsification, add subsequently concentration be 1% containing F68 pyrocarbonic acid diethyl ester (DEPC) aqueous solution in, again ultrasonic, then stirred at ambient temperature 1-2h, removing organic facies, obtains mPEG-PLGA-PLL-La-VEGFab/miRNA nanoparticle solution.Mainly concentrate between 10-300nm, especially based on the particle diameter of 100nm.
Water dispersion medium of the present invention comprises the various surfactants being suitable for preparing nanoparticle such as dextran 40, dextran 70, Pluronic F68, and its concentration is 0.1 ~ 10%.
Organic solvent of the present invention comprises the various organic solvents being suitable for preparing nanoparticle such as ethyl acetate, dichloromethane, acetone, ethanol.
Of the present invention pair of target cationic polymer nanoparticle may be used for preparing cancer therapy drug or the carrier as cancer therapy drug.
Antitumor drug of the present invention can be organic drug, water soluble drug or water-insoluble drug anticarcinogen, as comprised miRNA, siRNA, Chinese medicine monomer, ginsenoside and vascellum esoderma inhibin etc.Be particularly useful for preparing intravenous anti-cancer drug preparation.
MPEG-PLGA-PLL-La-VEGFab nanoparticle of the present invention by while target tumor histiocyte and tumor vessel come efficient specific delivery of gene medicine to tumor targets.
Preparation method of the present invention is easy, is suitable for large-scale production, is adapted to the antineoplastic agent nanoparticle drug-supplying system preparing cancer target especially.
Accompanying drawing illustrates:
Fig. 1. the mPEG-PLGA-PLL-LA-VEGFab nanoparticle grain size distribution of load microRNA-99amimics (miRNA)
Fig. 2. the mPEG-PLGA-PLL-LA-VEGFab nanoparticle transmission electron microscope picture of load miRNA
Fig. 3. the mPEG-PLGA-PLL-LA-VEGFab nanoparticle of load miRNA hatches the cytotoxicity figure after HepG2 hepatoma carcinoma cell 2h
Fig. 4. the mPEG-PLGA-PLL nanoparticle of the miRNA of (A) load fluorescent probe Cy5 labelling hatches the laser confocal microscope figure after HepG2 hepatoma carcinoma cell 2h with the mPEG-PLGA-PLL-LA-VEGFab nanoparticle of the miRNA of (B) load fluorescent probe Cy5 labelling
Fig. 5. (A) blank group, T suppression cell transition graph when the mPEG-PLGA-PLL-LA-VEGFab nanoparticle of (B) load miRNA hatches 24h.
Symbol description
Fig. 1: Size (d.nm) particle diameter is abscissa, and English Intensity (percent) intensity is vertical coordinate.Sizedistributionbyintensity represents the size distribution figure of nanoparticle intensity.
Fig. 2: the scale of transmission electron microscope picture is 200nm.
Fig. 3: nanoparticle concentration is abscissa, OD value is vertical coordinate.
Fig. 4 .A is the design sketch that non-targeted nanoparticle sends miRNA, and B has the design sketch that active targeting characteristic nanoparticle sends miRNA.
Fig. 5 .A is the blank group not doing any process, and B is the picture that lotus DNA loaded nanoparticle sends the migration of miRNA T suppression cell.
Detailed description of the invention:
Below with embodiment to the present invention's further instruction in addition, but do not limit content of the present invention.
Prepared by embodiment 1.mPEG-PLGA-PLL-LA-VEGFab carrier
.mPEG-PLGA-PLL-LA-VEGFab synthetic route
According to above-mentioned Macroscopic single crystal route, specifically describe as follows:
Wherein, mPEG-PLGA-PLL according to literature method synthesis (
peifengLiu, etal.AmPEG-PLGA-b-PLLcopolymercarrierforadriamycinandsiR NAdelivery.Biomaterials2012,33 (17): 4403-4412.).
(1) mPEG-PLGA of hydroxyl is synthesized: monomethyl ether-polyethylene glycol-(lactide coglycolide) (mPEG-PLGA) passes through D, L-lactide and Acetic acid, hydroxy-, bimol. cyclic ester ring-opening polymerisation obtain, and adopt stannous octoate as ring opening catalyst [35].Join in dry reaction test tube by 5gmPEG, 18.6gD, L-lactide and 6.4g Acetic acid, hydroxy-, bimol. cyclic ester, displacement nitrogen 3 times, 0.06g stannous octoate (0.2wt%mPEG, D, L-lactide and glycolide) is dissolved in 1mL anhydrous n-hexane and joins in reaction bulb.Vacuum drying 1 hour volatilization normal hexane, displacement nitrogen 3 times, vacuum state lower seal reaction bulb the last time.130 DEG C of heating 8h in oil bath, room temperature cools, and product is dissolved in chloroform, precipitates in a large amount of methanol, repeats precipitation for several times, filters and obtain 28.5g product, room temperature in vacuo kept dry.(2) mPEG-PLGA of synthesis containing Boc-L-Phe group: under temperature is-10 DEG C of conditions, 10gmPEG-PLGA, 1gBoc-L-Phe is dissolved in 80mL anhydrous methylene chloride, nitrogen protection, under 0 DEG C of condition, 0.8gDCC and 0.08gDMAP is added in reactant liquor, room temperature reaction 2 days, white precipitate in filtration under diminished pressure solution, gained solution saturated sodium bicarbonate washes twice (2 × 100mL), wash twice (2 × 100mL), anhydrous magnesium sulfate drying gained organic solution, filter, concentrated, white solid product is settled out in a large amount of ice methanol, filter, repeat precipitation and obtain 10.1g white product for several times, room temperature in vacuo kept dry.(3) mPEG-PLGA-Phe-NH is synthesized
2: end group is that amino mPEG-PLGA obtains by removing Boc-L-Phe blocking group tert-butoxy carboxyl in upper step product.Be that the mPEG-PLGA of Boc-L-Phe is dissolved in 70mL anhydrous methylene chloride by 6g end group, cooling solution to 0 DEG C, adds 15mL trifluoroacetic acid under nitrogen protection.Reaction 2h, vacuum rotary steam removes trifluoroacetic acid and dichloromethane, and residue is dissolved in chloroform again, and saturated sodium bicarbonate washes twice, washing twice, dried over sodium sulfate.Finally, product precipitates in a large amount of cooling methanol, and product is collected in pressure filtration, and repeat precipitation and obtain 4.8g product for several times, room temperature in vacuo is dry.(4) mPEG-PLGA-b-PLL (N is synthesized
ε-(Z)-
l-lysine): mPEG-PLGA-b-PLL (N
ε-(Z)-
l-lysine) being mainly amino mPEG-PLGA and NCA by end group, ring-opening polymerization realizes.The mPEG-PLGA-Phe-NH of NCA and 4g of 4g
2be dissolved in the dry chloroform of 80mL, join in dry flask.Nitrogen replaces 3 times, solution room temperature reaction 72h.After reaction terminates, revolve and steam evaporating liquid to 30mL, gained solution is placed in a large amount of chilled ethyl ether, precipitation obtains white product, filters, and repeat precipitation and obtain 6.8g polymer for several times, room temperature in vacuo is dry.(5) mPEG-PLGA-PLL is synthesized: 1g will walk the mPEG-PLGA-b-PLL (N of gained
ε-(Z)-
l-lysine) polymer joins (130 DEG C of dry 4h, nitrogen blast-cold) in dry reaction bottle, and then in reaction bulb, add 10mL hydrobromic acid/acetum dissolve polymer by syringe and form mud.Reaction bulb displacement nitrogen 3 times, reactant liquor room temperature reaction 1h.Finally, reactant liquor is joined precipitation in a large amount of ice ether and obtain the product mPEG-PLGA-PLL of 0.85g, collected by filtration, room temperature in vacuo is dry.(6) preparation of mPEG-PLGA-PLL-LA-VEGFab: get 1gmPEG-PLGA-PLL and be dissolved in dimethyl sulfoxine, add 100mgLA, 50mgVEGFab, 300mgEDC and 200mgNHS subsequently, stirring reaction 2-24h.Be the 3-96h that dialyses in the bag filter of 1000 as molecular retention amount by solution after reaction terminates, lyophilizing is subsequently preserved, and obtains the mPEG-PLGA-PLL-LA-VEGFab material that molecule is 30000.(6) preparation of mPEG-PLGA-PLL-LA-VEGFab: get 1gmPEG-PLGA-PLL and be dissolved in dimethyl sulfoxine, add 100mgLA, 50mgVEGFab, 300mgEDC and 200mgNHS subsequently, stirring reaction 2-24h.Be the 3-96h that dialyses in the bag filter of 1000 as molecular retention amount by solution after reaction terminates, lyophilizing is subsequently preserved, and obtains the mPEG-PLGA-PLL-LA-VEGFab material that molecule is 30000.
The preparation of the mPEG-PLGA-PLL-La-VEGFab/miRNA nanoparticle of embodiment 2. load microRNA-99amimics (miRNA)
Getting 4mg material mPEG-PLGA-PLL-La-VEGFab is dissolved in the mixed solution of dichloromethane or dichloromethane and acetone, add 2nmoLmiRNA, ultrasonic emulsification, add subsequently concentration be 1% containing F68 pyrocarbonic acid diethyl ester (DEPC) aqueous solution in, again ultrasonic, then stirred at ambient temperature 1-2h removes organic facies, obtains mPEG-PLGA-PLL-La-VEGFab/miRNA nanoparticle solution.As shown in Figure 1, nanoparticle has good size distribution to the result that nanoparticle is tested by laser particle analyzer, and particle diameter mainly concentrates between 10-300nm, especially based on the particle diameter of 100nm.As shown in Figure 2, the nanoparticle of preparation is rounded for transmission electron microscope results, has good dispersibility.
The cytotoxicity of the embodiment 3.mPEG-PLGA-PLL-La/VEGFab/miRNA grain of rice
HepG2 hepatoma carcinoma cell spreads 96 orifice plates, and the density of every porocyte is 1 × 10
5individual, at 37 DEG C, CO
2volume fraction is overnight incubation in the cell culture incubator of 5%.Joined by the nanoparticle of variable concentrations respectively (groups of cells only adding culture fluid is negative control group) in 96 orifice plates, each experiment condition arranges 5 multiple holes.Add MTT (5mg/mL) solution 20 μ L at cultivation 24h, continue to cultivate 4h, draw culture fluid afterwards, after adding DMSO100 μ L dissolving, test at microplate reader 490nm place and calculate the survival rate of cell.
As shown in Figure 3, compared with matched group, the OD value of the cell that nanoparticle is hatched, without obviously changing, shows that nanoparticle has good biocompatibility to experimental result.
Embodiment 4.mPEG-PLGA-PLL-La/VEGFab/miRNA nanoparticle laser confocal microscope figure
For ease of carrying out laser co-focusing observation, carrying out labelling miRNA with fluorescent probe Cy5 in experiment and studying.The coverslip of sterilizing is placed in 24 orifice plates, and HepG2 hepatoma carcinoma cell spreads 24 orifice plates, and the density of every porocyte is 2 × 10
5individual, 0.5mL trains liquid, at 37 DEG C, and CO
2volume fraction is overnight incubation in the cell culture incubator of 5%, after cell attachment, removes culture fluid.In different hole, add the culture fluid 0.2mL of mPEG-PLGA-PLL/miRNA nanoparticle and mPEG-PLGA-PLL-La-VEGFab/miRNA nanoparticle respectively, continue in 37 DEG C of cell culture incubators to cultivate 2h.Draw out culture fluid, add 4% paraformaldehyde and fix 20min, with PBS rinsing 3 times, each 5min, glycerol mounting, observes the distribution situation taking in nanoparticle in cell under laser confocal microscope.Lucifuge operation is carried out in above-mentioned experiment.
Experimental result as shown in Figure 4 B, compared with the mPEG-PLGA-PLL/miRNA nanoparticle without targeting (Fig. 4 A), the fluorescence intensity of the HepG2 tumor cell that mPEG-PLGA-PLL-La-VEGFab/miRNA nanoparticle is hatched obviously increases, and shows that mPEG-PLGA-PLL-La-VEGFab/miRNA nanoparticle can effectively improve the efficiency of sending miRNA.
Embodiment 5.mPEG-PLGA-PLL-La/VEGFab/miRNA T suppression cell transition graph
HepG2 hepatoma carcinoma cell spreads 6 orifice plates, and the density of every porocyte is 2.5 × 10
5individual, at 37 DEG C, CO
2volume fraction is overnight incubation in the cell culture incubator of 5%, after cell attachment, carry out cut with 10uL rifle head to take pictures, add in different hole respectively after mPEG-PLGA-PLL-La-VEGFab/microRNA nanoparticle 1mL cultivates 24h and take pictures again, observation of cell migration situation.Cellular control unit is left intact.
Experimental result as shown in Figure 5 B, compared with the cell migration of matched group (Fig. 5 A), distance between the HepG2 hepatoma carcinoma cell cut of mPEG-PLGA-PLL-La-VEGFab/microRNA nanoparticle process obviously increases, and shows that cell migration is suppressed effectively by nanoparticle.
Claims (10)
1. the selectively targeted tumor tissue cell of energy and tumor vascular pair of targeting methyl ether PEG-PLGA-polylysine-lactobionic acid-VEGF antibody mPEG-PLGA-PLL-LA-VEGFab cationic polymer, described polymer molecular weight is 2.0 × 10
3-10.0 × 10
6; Wherein the weight ratio of mPEG/PLGA is 1-50:50-100, in PLGA, the weight ratio of LA/GA is 1-100:1-100, the weight ratio of PLGA/PLL is 50-100:1-50, mPEG-PLGA-PLL, the weight ratio of LA and VEGFab is 1:0.0001-0.001:0.0001-0.001.
2. one kind as claimed in claim 1 by the nanoparticle of mPEG-PLGA-PLL-LA-VEGFab cationic polymer as carrier; it is characterized in that described nanoparticle is the nanoparticle of mPEG-PLGA-PLL-LA-VEGFab or the nanoparticle of mPEG-PLGA-PLL-LA-VEGFab/miRNA; particle size range is at 10-1000nm, and form is spherical.
3. nanoparticle as claimed in claim 2, is characterized in that described LA can a large amount of asialoglycoprotein receptor of existing of selectively targeted cancer cell surfaces, and can stable bond with it; Described VEGFab can selectively targeted other tumor neogenetic blood vessels endothelial factor VEGF, and specific binding with it.
4. the preparation method of a two target cationic polymer as claimed in claim 1, it is characterized in that at ambient temperature with in organic solvent, methyl ether PEG-PLGA-polylysine mPEG-PLGA-PLL and lactobionic acid LA, VEGF antibody VEGFab, carbodiimide EDC and N-hydroxy-succinamide NHS react 2-24h, reaction terminate after by solution as the 3-96h that dialyses in bag filter, lyophilizing; The weight ratio of described mPEG-PLGA-PLL, LA, VEGFab, EDC and NHS is 1:0.0001 ~ 0.001:0.0001 ~ 0.001:0.001 ~ 0.01:0.001 ~ 0.01.
5. the preparation method of two target cationic polymer as claimed in claim 4, is characterized in that described organic solvent is dimethyl sulfoxine, dichloromethane, chloroform or methanol; The molecular cut off of bag filter is 600-2000.
6. a preparation method for nanoparticle as claimed in claim 2, is characterized in that described two target cationic polymers to obtain nanoparticle by multi-emulsion method, coprecipitation, emulsion solvent diffusion method.
7. the preparation method of nanoparticle as claimed in claim 6, it is characterized in that described multi-emulsion method is dissolved in by mPEG-PLGA-PLL-La-VEGFab carrier in the organic solvent of ethyl acetate, dichloromethane or dichloromethane and acetone mixing, add pharmaceutical aqueous solution, ultrasonic or high pressure is newborn evenly obtains colostrum, add water dispersion medium again, after emulsifying obtains emulsion, stir or rotary evaporation removing organic facies. obtain mPEG-PLGA-PLL-La-VEGFab nanoparticle;
Described coprecipitation is dissolved in acetone by mPEG-PLGA-PLL-La-VEGFab carrier, is added drop-wise in the water dispersion medium of drug containing under stirring condition, stirs and wave most organic solvent, obtain the mPEG-PLGA-PLL-La-VEGFab nanoparticle of load medicine;
Described emulsion solvent diffusion method is dissolved in by mPEG-PLGA-PLL-La-VEGFab carrier in acetone/dichloromethane mixed solvent, join in the water dispersion medium of drug containing, the newborn even emulsifying of ultrasonic or high pressure, emulsion at room temperature waves most organic solvent, obtains the mPEG-PLGA-PLL-La-VEGFab nanoparticle of load medicine.
Or mPEG-PLGA-PLL-La-VEGFab is dissolved in the mixed solution of dichloromethane or dichloromethane and acetone, add miRNA, ultrasonic emulsification, add concentration be 1% containing F68 pyrocarbonic acid diethyl ester DEPC aqueous solution in, again ultrasonic, stirred at ambient temperature 1-2h, removing organic facies, obtains mPEG-PLGA-PLL-La-VEGFab/miRNA nanoparticle solution.
8. a target cationic polymer as claimed in claim 1 two is for the preparation of the carrier of Hepatoma therapy medicine.
9. a nanoparticle as claimed in claim 2 is for the preparation of Hepatoma therapy medicine.
10. apply as claimed in claim 8 or 9, it is characterized in that described Hepatoma therapy medicine is intravenous preparation.
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| CN110214145B (en) * | 2017-01-25 | 2023-04-14 | 四川大学 | CP-iRGD polypeptide, iDPP nanoparticle, drug-loaded compound and preparation method and application thereof |
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| CN114573706A (en) * | 2020-11-30 | 2022-06-03 | 中国科学院大连化学物理研究所 | Transporter coated with cross-linking agent for targeting chloroplast and application |
| CN114573706B (en) * | 2020-11-30 | 2024-03-08 | 中国科学院大连化学物理研究所 | Transport carrier coated with crosslinking agent for targeting chloroplast and application |
| CN114272370A (en) * | 2021-12-03 | 2022-04-05 | 中国人民解放军陆军军医大学第一附属医院 | MGaV nano-drug and application thereof |
| CN117982464A (en) * | 2024-01-26 | 2024-05-07 | 海南大学 | Nanoparticle as well as preparation method and application thereof |
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