CN105497902A - EGF (epidermal growth factor) ligand targeted nano drug-loaded system and anti-tumor treatment application thereof - Google Patents
EGF (epidermal growth factor) ligand targeted nano drug-loaded system and anti-tumor treatment application thereof Download PDFInfo
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Abstract
The invention discloses an EGF (epidermal growth factor) ligand targeted nano drug-loaded system and an anti-tumor treatment application thereof. According to the drug-loaded system, superparamagnetic nanoparticles are taken as magnetic nuclei, carboxymethyl chitosan is taken as a coating material, an anti-tumor drug is transferred to the coating layer, and the carboxymethyl chitosan surface is modified with an EGF. The EGF ligand targeted nano drug-loaded system has the characteristics of good biocompatibility, high stability, good slow release performance and specific target performance, so that the system has high targeted selectivity on tumor cells, can directionally release a drug to a tumor location, then plays roles of targeted inhibition and tumor cell killing and improves the bioavailability of the drug, and a new strategy is provided for targeted therapy of tumors; a preparation method of nano-carriers and the nano drug-loaded system is simple and convenient to operate, reaction conditions are mild, a prepared product can be taken as a wide carrier for the anti-tumor drug, and a new development direction is provided for study of the efficient anti-tumor drug targeted delivery carrier.
Description
Technical field
The present invention relates to biological nano medicine carrying material and drug targeting transport field, be specifically related to a kind of egf ligand body targeted nano medicine-carrying system and antineoplaston application thereof.
Background technology
Malignant tumor (cancer) is the first killer that harm humans is healthy, threaten human life, has that sickness rate is high, mortality rate is high, treatment is difficult and the feature such as easy recurrence.According to World Health Organization (WHO) up-to-date " report of world's cancer ", within 2012, there are 1,400 ten thousand newly-increased cases of cancers in the whole world, and number of cancer deaths reaches 8,200,000.The new cancer diagnosis case of China is 3,070,000, and account for 21.8% of whole world sum, number of cancer deaths about 2,200,000, account for global number of cancer deaths's 26.9%.And according to current pathogenesis of cancer trend, will than increasing by 50% now to the year two thousand twenty whole world cancer morbidity.Therefore, oncotherapy faces difficult task more, and current chemotherapy is as one of tumor essential therapeutic arsenals, owing to lacking the selectivity to tumor cell, while killing tumor cell, also destroy normal tissue cell, and then produce a series of toxic and side effects.Therefore, development is a kind of guides antitumor drug targeted to " focus " position and the pharmaceutical carrier improving target area drug level seems particularly urgent.
Along with nanotechnology deepening continuously in oncobiology research, magnetic nanoparticle (MNPs) is because having the features such as the superparamagnetism of good biocompatibility, hypotoxicity, the easy functionalization in surface and uniqueness, for tumor research provides a kind of novelty, has much the research material of prospect, using functional magnetic nano-particle as carrier, targeted antitumor drug become oncotherapy research focus.Carboxymethyl chitosan (CMC) is a kind of soluble derivative that chitosan generates after carboxylation reaction, has the characteristics such as good stability, tissue and blood compatibility, degradability and effective membrane penetrating.Due to it be a kind of containing active amino, carboxyl amphipathic degradability polysaccharide, can with multiple biological active matter coupling, thus become the preferred capsulating material of pharmaceutical carrier of new generation.By can improve the dispersion stabilization of carrier, the compatibility and water solublity at magnetic nanoparticle (MNPs) pan coating carboxymethyl chitosan (CMC).
Functional magnetic nano-particle can overcome as drug administration carrier the defect that convenient administration mode brings, as medicine makes target area reach desired concn very soon around along with carrier is transported to target area (tumor locus), therefore dosage can be reduced, reduce drug degradation, improve medicine stability and bioavailability, the circulation time in vivo of prolong drug, the slow release realizing medicine and controlled release etc., will the leading of oncotherapy be become.But still there is many technical bottleneck problems and wait to solve in this field at present: curative drug is faced with how by special and be effectively transported to the difficult problem of target (tumor) cell; Can carrier effectively be transported to appointed part, proceed to target cell and play a role in kytoplasm by safety of medicine; How to improve carrier surface character, to avoid carrier by reticuloendothelial system phagocytics such as liver, spleen, lung, bone marrow; How to improve the drug loading etc. of carrier.For the problems referred to above, targeting-functionalization magnetic nano-carrier is that the targeting transport of antitumor drug provides a new direction.
Summary of the invention
The object of the invention is to provide the problem that the object of the invention is to in the urgent need to address in current antitumor drug targeted difficulty, easily degraded and the oncotherapy such as circulation time in vivo is short, builds a kind of Nano medication delivery system with ligand specificity's target function.
Technical scheme of the present invention is: EGF part targeted nano medicine-carrying system, and medicine-carried system, using superparamagnetic nano particle as magnetic core, is capsulating material with carboxymethyl chitosan, wherein loads antitumor drug, and at its finishing epidermal growth factor EGF.
Further, described magnetic core is the ferroferric oxide nano granules of superparamagnetic performance.
Further, described antitumor drug is vincristine VCR.
The preparation method of EGF part targeted nano medicine-carrying system, comprises the following steps:
1. the preparation of magnetic core: utilize partial reduction liquor ferri trichloridi coprecipitation to prepare ferroferric oxide nano granules;
2. magnetic nuclear membrane and medicine load: prepare medicine-CMC/MNPs by reverse microemulsion process at ferroferric oxide nano granules pan coating carboxymethyl chitosan and loading antitumor drug;
3. EGF modified medicaments-CMC/MNPs: adopt the method for EDC activated carboxyl coupling epidermal growth factor EGF to prepare EGF-medicine-CMC/MNPs.
Further, the preparation method of EGF part targeted nano medicine-carrying system, comprises the following steps:
1. partial reduction ferric chloride (FeCl is utilized
3) solution coprecipitation, prepare carrier core ferroferric oxide nano granules solution under magnetic agitation condition, deoxygenated water washing, vacuum drying, grinding are filtered and obtain ferroferric oxide nano granules;
2. adopt the carboxymethyl chitosan (CMC) of biocompatibility as magnetic nuclear membrane material, utilize reverse microemulsion process under the condition of nitrogen protection, mechanical agitation is carried out bag quilt and is loaded antitumor drug, products therefrom carries out Magneto separate washing under the condition of externally-applied magnetic field, vacuum drying, collects and is medicine-CMC/MNPs;
3. react and carry out in MES solution, after adding EDC mixing, add EGF, oscillating reactions, products therefrom PBS Magneto separate washing, obtains EGF-medicine-CMC/MNPs.
Further, step concrete grammar is 3.:
By medicine-CMC/MNPs ultrasonic disperse in MES solution, pH4.5-7.4, after adding 1mg/mLEDC mixing, add 0.5-2mg/mL epidermal growth factor, high speed oscillating reactions 2h, products therefrom phosphate buffered saline(PBS) (PBS) Magneto separate washing, prepares the medicine carrying complex EGF-medicine-CMC/MNPs of egf ligand body targeting.
Active targeting magnetic nanoparticle is combined by the specific part of its surperficial coupling (or antibody) receptor-specific ground corresponding to target cells, promote that target cell is to the absorption of medicine, avoid engulfing by reticuloendothelial system macrophages such as liver, spleen, lung, bone marrow, greatly improve the specificity of carrier, targeting and selectivity, improve the concentration of medicine in tumor tissues.
EGF-R ELISA (EGFR) is the multi-functional glycoprotein that proto-oncogene c-erbBl expresses on cell membrane, can be combined with epidermal growth factor (EGF) ligand specificity.In the tumor tissues of different differentiation degree, its expression increases with malignancy of tumor rank and increases, this EGF-R ELISA differential expression makes EGF-R ELISA become a kind of potential tumor targeted molecular mark, therefore, our option table skin growth factor part is as the targeted molecular modifying magnetic nano particle, the specific binding on cell membrane by EGF-R ELISA and its part epidermal growth factor, build the magnetic nano particle of egf ligand body active targeting as antitumor drug delivery vehicles, improve the targeting of antitumor drug, selectivity and aggregation.
The present invention compared with prior art tool has the following advantages:
Epidermal growth factor (EGF) targeted nano carrier prepared by the present invention, has the targeting selectivity of height, compares non-targeted nanometer medicine-carried system, significantly improve the bioavailability of medicine, reduces the toxic and side effects of normal tissue cell.
The preparation method of nano-carrier of the present invention and medicine-carried system is easy and simple to handle, reaction condition is gentle, prepares the extensive carrier that product can be used as antitumor drug, for efficient antitumor drug targeted study on the carrier provides a kind of new developing direction.
Accompanying drawing explanation
Fig. 1 is structure and the targeted inhibition glioma propagation schematic diagram of EGF coupled to Nano medicine-carried system.
Transmission electron microscope (TEM) figure of Fig. 2 nanometer medicine-carried system constructed by embodiment 1.
Fig. 3 is infrared spectrum (FT-IR) figure of embodiment 1EGF targeted nano medicine-carrying system.
Fig. 4 is the magnetic response figure of embodiment 2EGF targeted nano medicine-carrying system.
Fig. 5 is that embodiment 3EGF targeted nano medicine-carrying system suppresses U251 cell proliferation.
Fig. 6 is that embodiment 4EGF targeted nano medicine-carrying system causes U251 cytoactive to reduce.
Fig. 7 is that embodiment 5EGF targeted nano medicine-carrying system causes U251 cytoskeleton injury.
Fig. 8 is that embodiment 6EGF targeted nano medicine-carrying system causes U251 apoptosis.
Fig. 9 is embodiment 7EGF targeted nano medicine-carrying system retardance U251 cell cycle progression.
Detailed description of the invention
Below in conjunction with the drawings and specific embodiments, the present invention is described in detail.
Embodiment 1
The structure schematic diagram of EGF part targeted nano medicine-carrying system is see a Fig. 1, and its concrete preparation process is as follows:
(1) preparation of magnetic core and superparamagnetic nano particle (MNPs): draw 10.5mL deionized water in three-neck flask, add the FeCl of 3mL2mol/L
3solution, starts magnetic agitation with suitable speed; At the uniform velocity drip the Na of 2mL
2sO
3; When color is reduced to again yellow by rufous, start the NH slowly dripping 80mL
3.H
2o solution, and vigorous stirring, black precipitate generates rapidly; Continue to stir 40min, to ensure to react completely; With deoxygenated water washing black precipitate 3 times, vacuum drying under 45 DEG C of conditions, carefully grinding is filtered and obtains Fe
3o
4powder.
(2) adopt reverse microemulsion legal system for medicine-CMC/MNPs: to get appropriate antitumor drug, CMC and MNPs(mass ratio 2:1) be dissolved in the deionized water (DI) of 5mL, at nitrogen (N
2) under protection, supersound process 30min, forms suspension.Meanwhile, in there-necked flask, add 50mL liquid paraffin and sorbester p17, at nitrogen (N
2) protecting lower 55 ° of water-baths, 600rpm is stirred to milk; Then above-mentioned suspension is dropwise dropped in there-necked flask, high-speed stirred 2h, then stirring at low speed 10h, form water-in-oil microemulsion, under the condition of externally-applied magnetic field, carry out Magneto separate precipitation, with petroleum ether, isopropyl alcohol replaces washing three times, finally as in petroleum ether, and vacuum drying.Collection is medicine-CMC/MNPs powder.
(3) method of EDC activated carboxyl coupling EGF is adopted to prepare EGF-medicine-CMC/MNPs: to get 5mgCMC/MNPs or medicine-CMC/MNPs powder, add in 1.5mLEP pipe after ultra-vioket radiation 10min, add 0.5mLMES solution again, ultrasonic 5min, powder is fully dissolved; Meanwhile, add 20 μ LEDC in above-mentioned CMC/MNPs solution, after mixing, add the EGF of 5 μ L, be positioned over ZDY-1 type concussion instrument, shake reaction at a high speed.With the washing of PBS Magneto separate, be finally placed in PBS, obtain EGF-medicine-CMC/MNPs.
Antitumor drug in above-described embodiment is vincristine (VCR), and the part targeted drug magnetic nano particle complex of preparation is EGF-VCR-CMC/MNPs.
Adopt transmission electron microscope (TEM) to characterize obtained support C MC/MNPs, can be observed the phenomenon of Fig. 2.Fig. 2 shows that the pattern of sample is spherical or oval nano-particle, and mean diameter is ± 60nm.
Obtained medicine carrying EGF-VCR-CMC/MNPs is adopted the composition of infrared spectrum characterization product, can be observed the result of Fig. 3.Fig. 3 shows successfully to prepare superparamagnetism carrier core MNPs(Fe-O characteristic absorption peak at 567.7cm
-1); Carboxymethyl chitosan (CMC) successfully wraps by superparamagnetism carrier core MNPs, and antitumor drug vincristine (VCR) successfully loads and the success of EGF targeting modification.
Magnetic response Performance Detection: the EGF-VCR-CMC/MNPs of preparation is scattered in DI water, under externally-applied magnetic field and naturalness, at regular intervals (0,10,20,30,40,50,60min) utilize ultraviolet-visible spectrophotometer to measure the change of its 570nm place light transmittance.By the change of transmittance values, observe nano-particle magnetic response ability.Fig. 4 is the magnetic response figure of EGF nanometer medicine-carried system EGF-VCR-CMC/MNPs under naturalness and magnetic field state, as can be seen from the figure, EGF-VCR-CMC/MNPs medicine-carried system has good dispersibility in its natural state, puts into magnetic field and has good magnetic responsiveness.
cell culture experiments
Glioma U251 cell is at 5%CO
2cellar culture is carried out with using the RPMI-1640 cell culture fluid containing 10% new-born calf serum, 100U/mL penicillin and 100 μ g/mL streptomycins under 37 DEG C of environment.When Growth of Cells reach 90% converge time, carry out routine passage in 1:3 ratio, to ensure that cell is in exponential phase.The cell of exponential phase is in, after cell suspension counting, according to 1x10 with pancreatin process
4density be seeded to 96 well culture plates and cultivate.Setup Experiments non-targeted CMC/MNPs group, targeting EGF-CMC/MNPs group, non-targeted VCR-CMC/MNPs and targeting EGF-VCR-CMC/MNPs dosing group, often group arranges 3 repetitions.After cultivating 24h, add each concentration medicine-carried system (0,50,100,200,400,800 μ g/mL).After process 24h, liquid in sucking-off hole, after washing twice, adds the MTT that final concentration is 0.5mg/mL with PBS, continue to hatch 4h in incubator after, add the dimethyl sulfoxide (DMSO) of 150 μ L, after lucifuge oscillation incubation 15min, with the absorbance at microplate spectrophotometric measurement 570nm place.
Mtt assay is utilized to detect various nano-carrier process to the impact of U251 ability of cell proliferation.As can be seen from Figure 5, compared with untreated contrast, within the scope of test concentrations, the multiplication capacity of CMC/MNPs and EGF-CMC/MNPs vehicle group to U251 cell is and has an impact; VCR-CMC/MNPs and EGF-VCR-CMC/MNPs group all significantly reduces the multiplication capacity of U251 cell, and the multiplication capacity of EGF-VCR-CMC/MNPs group on U251 cell creates and more significantly affect.
cell activation assay is tested
0.25% tryptic digestive juice 37 DEG C condition digestion is in the U251 cell 3min of exponential phase; The centrifugal 5min collecting cell of 1000rpm, counting, stand-by.Cell is pressed 2x10
4the density of cells/well is seeded to 24 orifice plates and cultivates.Arrange matched group (untreated U251 cell), EGF targeting and non-targeted dosing group in experiment, often group arranges 3 repetitions.Each concentration medicine-carried system (0,50,100,200,400,800 μ g/mL) is added after cultivating 48h, cell is after process 24h, add propidium iodide (PI) solution that diacetic acid fluorescein (FDA) solution that final concentration is 1 μ g/mL and final concentration are 20 μ g/mL respectively, 10min is hatched under room temperature condition, PBS buffer solution, the activity of observation of cell under fluorescence microscope.
The two dye method of fluorescent dye FDA/PI is utilized to analyze non-targeted VCR-CMC/MNPs and targeting EGF-VCR-CMC/MNPs process to the impact of U251 activity.After the two dye of FDA/PI, active good cell is dyed green by FDA, and dead cell or damaged cell are then dyed redness by PI.As can be seen from Figure 6, compared with untreated U251 cell, targeting EGF-VCR-CMC/MNPs process is obviously better than non-targeted group of VCR-CMC/MNPs to the impact of U251 cytoactive.
apoptosis test experience
Collect the U251 glioma cell being in exponential phase, according to 1x10
6density cell is seeded in six orifice plates, cultivate after 48h, add VCR-CMC/MNPs and EGF-VCR-CMC/MNPs of variable concentrations respectively, after continuing to cultivate 12h, collecting cell.Wash twice with PBS, cell is placed in 1.5mLEP pipe, the centrifugal 5min of 1000r/min.Then, add the CTAB of 500 μ L in each EP pipe, wherein adding beta-mercaptoethanol to final volume in CTAB is 0.1%, then adds the RNase that final concentration is 20 μ g/mL, and 65 ° of water-bath 30min, jiggle every 5min; Use phenol-chloroform-isoamyl alcohol extraction again, collect supernatant; Add isopyknic dehydrated alcohol ,-20 DEG C of precipitation 2h; Centrifugal 5min in refrigerated centrifuge; Abandoning supernatant, add the pre-cooled ethanol of 500 μ L75%, centrifugal, supernatant discards, and is inverted in absorbent paper by EP pipe, makes ethanol Ex-all; Often manage the TE adding 20 μ L again to dissolve; The sepharose electrophoresis of 1%.After electrophoresis, with gel imaging system, observe DNA band.
DNAladder method is utilized to detect VCR-CMC/MNPs and EGF-VCR-CMC/MNPs process to the apoptotic impact of U251.As can be seen from Figure 7, along with the continuous rising of nanometer system concentration, DNAladder is more obvious, and the DNAladder of the U251 cell of EGF-VCR-CMC/MNPs process is more obvious, and this illustrates that targeting EGF-VCR-CMC/MNPs process can cause more U251 apoptosis.
with the phalloidin of rhodamine labelling, detect the form of U251 cell after non-targeted group of VCR-CMC/MNPs and targeting group EGF-VCR-CMC/MNPs process and the structure of actin cytoskeleton F-actin
Cell is seeded in 24 orifice plates, after cultivating 24h, agent-feeding treatment; The culture medium being added with medicine discards by process 24h, uses PBS rinsing; With the isovaleral fixed cell 10-15min of 2.5%; PBS rinsing twice, the TritonX-100 adding 0.5% changes cell 10-15min thoroughly; With PBS rinsing twice, add the bad peptide working solution of Phallus rugulosus Fisch. of 200 μ L rhodamine labellings, hatch 30min; PBS rinsing three times; Nucleus redyed by DAPI dyestuff; With PBS rinsing cell twice, inverted fluorescence microscope is observed and is taken a picture.
Phalloidin is a kind of polypeptide protein extracted from poisonous mushroom, and its effect is stable Subfilament Structure.Phalloidin and F-actin have very high affinity, can specific binding with it, and its combination is very stable.Fig. 8 shows compared with untreated matched group, VCR-CMC/MNPs and EGF-VCR-CMC/MNPs all can cause the change of U251 cellular morphology, produce damaging action, and targeting group is more obvious to the damage of U251 cytoskeleton to cytoskeleton.
Flow cytometer process is on the impact in U251 cycle: first gather in the crops variable concentrations VCR-CMC/MNPs and EGF-VCR-CMC/MNPs(0,50,100,200,400,800 μ g/mL) cell after 12h, and be separated into cell suspension, PBS buffer solution three times also, after counting, gets 1x10
6the cell suspension of individual cell is resuspended in 500 μ LPBS buffer; 70% ethanol slowly adding 1mL pre-cooling, to above-mentioned cell suspension, mixes latter 4 DEG C and spends the night fixing; Discard fixative by centrifugal for cell suspension, be resuspended in 1mLPBS buffer, the centrifugal 5min of 1000r/min, discards PBS buffer; Add 1mLPI dye liquor (50 μ g/mL), 4 DEG C of lucifuges hatch 1h; Flow cytomery cell cycle distribution, then uses CellQuest software (BDBiosciences company) to analyze.Untreated U251 cell is set in test for contrast.
Research and utilization PI dyes, the flow cytometry analysis impact of nano-carrier process on U251 cell cycle distribution.As can be seen from Figure 9, compared with untreated contrast, along with the increase of nano-carrier concentration, G1 phase cell proportion obviously increases, and this illustrates that the propagation of U251 cell is arrested in the G1 phase.
The above embodiment only have expressed the detailed description of the invention of the application, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the application's protection domain.It should be pointed out that for the person of ordinary skill of the art, under the prerequisite not departing from technical scheme design, can also make some distortion and improvement, these all belong to the protection domain of the application.
Claims (7)
1.EGF part targeted nano medicine-carrying system, is characterized in that, medicine-carried system, using superparamagnetic nano particle as magnetic core, is capsulating material with carboxymethyl chitosan, wherein loads antitumor drug, and at its finishing epidermal growth factor EGF.
2. EGF part targeted nano medicine-carrying system according to claim 1, is characterized in that, described magnetic core is the ferroferric oxide nano granules with unique superparamagnetic performance.
3. EGF part targeted nano medicine-carrying system according to claim 1, it is characterized in that, described antitumor drug is vincristine VCR.
4. the preparation method of the EGF part targeted nano medicine-carrying system according to any one of claim 1-3, is characterized in that, comprise the following steps:
1. the preparation of magnetic core: utilize partial reduction liquor ferri trichloridi coprecipitation to prepare ferroferric oxide nano granules;
2. magnetic nuclear membrane and medicine load: by reverse microemulsion process at ferroferric oxide nano granules pan coating carboxymethyl chitosan and loading antitumor drug, prepare medicine-CMC/MNPs;
3. EGF modified medicaments-CMC/MNPs: adopt the method for EDC activated carboxyl coupling epidermal growth factor EGF to prepare EGF-medicine-CMC/MNPs.
5. the preparation method of EGF part targeted nano medicine-carrying system according to claim 4, is characterized in that, comprise the following steps:
1. partial reduction ferric chloride (FeCl is utilized
3) solution coprecipitation, prepare carrier core ferroferric oxide nano granules solution under magnetic agitation condition, deoxygenated water washing, vacuum drying, grinding are filtered and obtain ferroferric oxide nano granules;
2. adopt the carboxymethyl chitosan (CMC) of biocompatibility as magnetic nuclear membrane material, utilize reverse microemulsion process under the condition of nitrogen protection, mechanical agitation is carried out bag quilt and is loaded antitumor drug, products therefrom carries out Magneto separate washing under the condition of externally-applied magnetic field, vacuum drying, collects and is medicine-CMC/MNPs;
3. react and carry out in MES solution, after adding EDC mixing, add EGF, oscillating reactions, products therefrom PBS Magneto separate washing, obtains EGF-medicine-CMC/MNPs.
6. the preparation method of the EGF part targeted nano medicine-carrying system according to claim 4 or 5, it is characterized in that, step concrete grammar is 3.:
By medicine-CMC/MNPs ultrasonic disperse in MES solution, pH4.5-7.4, after adding 1mg/mLEDC mixing, add 0.5-2mg/mL epidermal growth factor, high speed oscillating reactions 2h, products therefrom phosphate buffered saline(PBS) (PBS) Magneto separate washing, prepares the medicine carrying complex EGF-medicine-CMC/MNPs of egf ligand body targeting.
7. the application of a kind of egf ligand body targeted nano medicine-carrying system in oncotherapy according to any one of claim 1-3.
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|---|---|---|---|---|
| CN112089849A (en) * | 2020-07-22 | 2020-12-18 | 首都医科大学附属北京天坛医院 | Therapeutic gene delivery vector and delivery system for targeting glioma |
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| CN102961345A (en) * | 2012-11-20 | 2013-03-13 | 桂林电子科技大学 | Method for preparing rapamycin/magnetic carboxymethyl chitosan nano drug-loaded microspheres |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112089849A (en) * | 2020-07-22 | 2020-12-18 | 首都医科大学附属北京天坛医院 | Therapeutic gene delivery vector and delivery system for targeting glioma |
| CN112089849B (en) * | 2020-07-22 | 2022-06-21 | 首都医科大学附属北京天坛医院 | Therapeutic gene delivery vector and delivery system for targeting glioma |
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