CN105031648A - Novel targeting nanoparticle as well as preparation method and application thereof - Google Patents
Novel targeting nanoparticle as well as preparation method and application thereof Download PDFInfo
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- CN105031648A CN105031648A CN201510528034.2A CN201510528034A CN105031648A CN 105031648 A CN105031648 A CN 105031648A CN 201510528034 A CN201510528034 A CN 201510528034A CN 105031648 A CN105031648 A CN 105031648A
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Abstract
The invention discloses a novel targeting nanoparticle as well as a preparation method and an application thereof, and belongs to the technical field of biological medicines. By a folate-targeted biodegradable positive electron polymer carrier PEI-CyD-FA (H1), double-chain DNA molecule Dbait (32bp) in a small fragment hairpin structure is encapsulated into a nanoparticle H1/Dbait, so that the radiotherapy sensitization effect is effectively realized. The nanoparticle has the advantages of low cytotoxicity, high transfection efficiency, high stability and the like, and is convenient to prepare; the targeted nanoparticle has relatively high transfection efficiency, relatively high stability and relatively low cytotoxicity; the radiosensitivity of tumor cells can be obviously increased; the tumor growth of mice with tumors is effectively inhibited; the survival time of the mice with tumors is prolonged; and the novel targeting nanoparticle has a significant effect on tumor treatment.
Description
Technical field
The present invention relates to novel targeted nanoparticle and the study on its developing thereof of load small fragment hair clip spline structure DNA molecular, belong to biomedicine technical field.
Background technology
Radiotherapy utilizes one or more ionizing radiation to treat malignant tumor or some benign lesions, and very little to the damage of normal surrounding tissue, become the Main Means for the treatment of malignant tumor at present.Bergonie and Tribondean proposed the law about radiosensitivity in 1906.Radiosensitivity refers to radiation effect, and the sensitivity of tumour radiotherapy depends primarily on their intrinsic sensitivity, tissue-derived, differentiation degree, general type, anemia, locally concurrent infection etc.Different tumors can be divided into radiation-sensitive according to the effect of radiotherapy tumor, radiate the tumor of medium sensitivity and radiation opposing.For the tumor of radiation opposing, permissive attitude or the refusal taked was treated more in the past.Therefore, utilize various supplementary means to reduce the radiation opposing of tumor, improve its radiosensitivity, alleviate simultaneously or avoid the radiation damage of normal structure as far as possible, become the study hotspot of radiotherapist.Along with going deep into of studying oncomolecularbiology, the concept of radiosensitizer expands to one from traditional anoxic cell sensitizer and contains many-sided complicated field, comprises cell micro-environment, DNA damage, cell cycle regulating etc.
In recent years, Institut Curie of radiation institute of country of France Dutreix reports a kind of Novel radioactive sensitizer Dbait.Dbait is one section of double chain DNA molecule with hair clip spline structure, is so small to have only tens bases.The mechanism of action of radiosensitizer conventional is at present suppress the single target spot in DNA damage repair pathways, and Dbait molecule has unique mechanism of action, Dbait molecule targeting can suppress one of main repair pathways of DNA damage---and non-homologous end joining repairs the formation of repairing focus in whole approach, improves the sensitivity of tumor to radiation by improving tumor cell sublethal damage.The simple application of Dbait molecule, without tumor inhibition effect, only has and combines could produce sensitization with therapeutic modalities such as the radiation causing DNA damage, chemotherapeutics.How targeting, efficiently transduction Dbait molecule are in tumor cell, and realize the difficult point that its radiosensitizing effect is current research.
Utilize carrier by exogenous gene accurately, efficient, import in host cell targeting, be the key technology of therapy of tumor.In genetic engineering, conventional artificial constructed plasmid is as genophore.Plasmid is the hereditary unit that can independently copy outside chromosome, comprises Deoxydization nucleotide (DNA) molecule in Eukaryotic organelle and bacterial cell beyond chromosome.After plamid vector construction success, imported in target cell by rotaring dyeing technology, it expresses the impact by conditions such as plasmid size, the amount proceeding to plasmid, cell types, and routine is used for engineered plasmid length and is mostly many kilobases to more than (bp).But, at present for how targeting, efficiently small fragment DNA molecular (length <50bp) transfection is imported host cell research still not thorough.
Summary of the invention
In order to overcome above-mentioned the deficiencies in the prior art part, the invention provides a kind of novel targeted nanoparticle and its production and use, use folate-targeted, biodegradable positron polymer support PEI-CyD-FA (H1), the double chain DNA molecule Dbait of small fragment hair clip spline structure is packaged into nanoparticle H1/Dbait, thus effectively realizes its Apoptosis.This nanoparticle has the advantages such as low cytotoxicity, high transfection efficiency, high stability, and easy to prepare.
The present invention is achieved through the following technical solutions: a kind of novel targeted nanoparticle, and described nanoparticle is made up of the double chain DNA molecule of folate-targeted, biodegradable positron polymer support PEI-CyD-FA (H1) load small fragment hair clip spline structure.The double chain DNA molecule <50bp of described small fragment hair clip spline structure.
Further, the particle diameter of described nanoparticle is 100 ~ 200 nanometers, and Zeta potential is 10mV ~ 20mV.
The preparation method of described novel targeted nanoparticle, comprises the following steps:
(1) getting positron polymer support PEI-CyD-FA (H1) is dissolved in distilled water, obtains solution 1, and room temperature, lucifuge leave standstill 5 minutes;
(2) the double chain DNA molecule Dbait getting small fragment hair clip spline structure is dissolved in distilled water, obtains solution 2, and room temperature leaves standstill 5 minutes;
(3) instill in solution 2 by solution 1 under lucifuge, vortex oscillation, continue vortex oscillation 15 seconds, solution 1 and solution 2 are fully mixed, and lucifuge, room temperature leave standstill and obtain novel targeted nanoparticle H1/Dbait after 20 minutes, namely can be used for follow-up related experiment.
Described novel targeted nanoparticle is used for the purposes of tumour radiotherapy.
Further, described tumor is carcinoma of prostate or colorectal cancer or hepatocarcinoma or pulmonary carcinoma or melanoma or breast carcinoma or ovarian cancer or cancer of pancreas or the esophageal carcinoma or gastric cancer or uterus carcinoma or laryngeal carcinoma or thyroid carcinoma.
The invention has the beneficial effects as follows: the invention provides a kind of structure of novel targeted nanoparticle and the application in tumour radiotherapy thereof, through experimental verification, this targeted nano-particle has higher transfection efficiency, higher stability, lower cytotoxicity, the radiosensitivity of tumor cell can be significantly improved, and effectively suppress the tumor growth of mice with tumor, the life span of prolongation mice with tumor, in oncotherapy, there is remarkable result.
Accompanying drawing explanation
The present invention is further described according to drawings and embodiments below.
Fig. 1 .1. granularity/Zeta potential analyzer detects the droplet measurement figure of novel targeted nanoparticle.
Fig. 1 .2. granularity/Zeta potential analyzer detects the particle size results cartogram of novel targeted nanoparticle.
Fig. 1 .3. granularity/Zeta potential analyzer detects the Zeta potential result cartogram of novel targeted nanoparticle.
Fig. 2. agarose gel electrophoresis detects stability and the encapsulation ratio result figure of novel targeted nanoparticle.
Fig. 3. the transfection efficiency result figure of the novel targeted nanoparticle of fluorescence microscopy Microscopic observation in Prostatic cancer cell lines PC3.
The cytotoxicity result figure of the novel targeted nanoparticle that Fig. 4 .MTT method detects.
Fig. 5 .1. colony formation detects the result figure that novel targeted nanoparticle affects Prostatic cancer cell lines PC3 clonality.
Fig. 5 .2. colony formation matching one-hit multitarget model and Linear quadratic model.
The novel targeted nanoparticle of Fig. 5 .3. is to Prostatic cancer cell lines PC3 radiation sensitizing effect relevant parameter statistical table.
Fig. 6 .1.WesternBlot detects the result figure of novel targeted nanoparticle on DNA damage label γ-H2AX expression impact in Prostatic cancer cell lines PC3 cell.
Fig. 6 .2.WesternBlot detects the gray analysis cartogram of novel targeted nanoparticle on DNA damage label γ-H2AX expression impact in Prostatic cancer cell lines PC3 cell.
The novel targeted nanoparticle of Fig. 7 .1. immuno-fluorescence assay is to DNA damage label γ-H in Prostatic cancer cell lines PC3 cell
2the result figure of AX expression impact.
The novel targeted nanoparticle of Fig. 7 .2. immuno-fluorescence assay is to DNA damage label γ-H in Prostatic cancer cell lines PC3 cell
2the luciferase expression amount cartogram of AX expression impact.
Fig. 8 .WesternBlot detects the result figure of novel targeted nanoparticle on DNA damage response protein developed by molecule level impact in Prostatic cancer cell lines PC3 cell.
Fig. 9. Immunohistochemical Method detects novel targeted nanoparticle to DNA damage label γ-H in PC3 tumor tissues
2the result figure of AX expression impact.
Figure 10. Immunohistochemical Method detects the result figure of novel targeted nanoparticle on DNA damage response protein DNA-PKcs expression impact in PC3 tumor tissues.
Figure 11 .WesternBlot detects the result figure of novel targeted nanoparticle on DNA damage response protein developed by molecule level impact in PC3 tumor tissues.
Figure 12. the result figure that novel targeted nanoparticle affects the tumor volume growth of mice with tumor.
Figure 13. novel targeted nanoparticle is on the result figure of mice with tumor impact life cycle.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is further described.
A kind of novel targeted nanoparticle, described nanoparticle is made up of the double chain DNA molecule of folate-targeted, biodegradable positron polymer support PEI-CyD-FA (H1) load small fragment hair clip spline structure; The double chain DNA molecule of described small fragment hair clip spline structure is Novel radioactive sensitizer Dbait molecule, and its size is 32bp.
Further, the particle diameter of described nanoparticle is 100 ~ 200 nanometers, and Zeta potential is 10mV ~ 20mV.
The preparation method of described novel targeted nanoparticle, comprises the following steps:
(1) getting positron polymer support PEI-CyD-FA (H1) is dissolved in distilled water, obtains solution 1, and room temperature, lucifuge leave standstill 5 minutes;
(2) the double chain DNA molecule Dbait getting small fragment hair clip spline structure is dissolved in distilled water, obtains solution 2, and room temperature leaves standstill 5 minutes;
(3) instill in solution 2 by solution 1 under lucifuge, vortex oscillation, continue vortex oscillation 15 seconds, solution 1 and solution 2 are fully mixed, and lucifuge, room temperature leave standstill and obtain novel targeted nanoparticle H1/Dbait after 20 minutes, namely can be used for follow-up related experiment.
Described novel targeted nanoparticle is used for the purposes of tumour radiotherapy.
Further, described tumor is carcinoma of prostate or colorectal cancer or hepatocarcinoma or pulmonary carcinoma or melanoma or breast carcinoma or ovarian cancer or cancer of pancreas or the esophageal carcinoma or gastric cancer or uterus carcinoma or laryngeal carcinoma or thyroid carcinoma.
In preferred embodiment of the present invention, select castration-resistant prostate cancer.
Human carcinoma of prostate cell line PC3 cell is purchased from Chinese Academy of Sciences's Shanghai cell biological institute.PC3 cell culture in containing 100 μ g/ml streptomycins, 100U/ml penicillin and 10% hyclone RPMI-1640 culture medium in, in 37 DEG C, 5%CO
2cultivate in incubator.
Dbait synthesizes in Nanjing Genscript Biotechnology Co., Ltd..
Particle diameter and the Zeta potential of the novel targeted nanoparticle of embodiment 1. detect
Get the H1/Dbait mixed liquor of freshly prepared different N/P ratio (1:1,6:1,12:1,18:1,24:1), adopt particle diameter/Zeta potential analyzer, the light scattering particle diameter of H1/Dbait nanoparticle, Zeta potential value are measured.
Stability and the encapsulation ratio of the novel targeted nanoparticle of embodiment 2. detect
Get the H1/Dbait mixed liquor of freshly prepared different N/P ratio (1:1,6:1,12:1,18:1,24:1), adopt agarose gel electrophoresis to detect the stability of novel targeted nanoparticle H1/Dbait and encapsulation ratio.The concrete steps of agarose gel electrophoresis are as follows:
(1) take 0.5g agarose and be placed in conical flask, add 50ml1 × TAE liquid, bottleneck back-off small beaker, microwave-oven-heating melts completely to agarose, seethes with excitement 3 times;
(2) when agarose gel liquid is cooled to 60 DEG C, add 5ul nucleic acid dye, pour into after shaking up in device glue groove for subsequent use, make coagulant liquid evenly be laid on glue groove bottom.Room temperature leaves standstill solidifies completely to gel, vertically pulls up the stripping fork in glue groove, is inserted by gel in electrophoresis tank, adds 1 × TAE electrophoretic buffer to not having gel 1-2mm.
(3), after mixing H1/Dbait mixed liquor and DNA sample-loading buffer, each sample is added in sample well respectively.
(4) electrophoresis immediately after application of sample, voltage 120V, 30 minutes time.
(5), after electrophoresis terminates, taking-up gel is placed in gel imaging system takes pictures and preserves.
The transfection efficiency of novel targeted nanoparticle to Prostatic cancer cell lines PC3 cell is observed under embodiment 3. inverted fluorescence microscope
(1) the Dbait molecule of Nanjing Genscript Biotechnology Co., Ltd.'s synthesis 5-FAM labelling, for fluoroscopic examination.
(2) the PC3 cell being in exponential phase is seeded in 6 orifice plates, 3 × 10
5/ hole, with the RPMI1640 culture medium cellar culture containing 10% hyclone, 100 μ g/ml streptomycins, 100U/ml penicillin.When cell fusion degree reaches 70%-80%, replace medium to antibiotic-free, RPMI1640 culture medium containing 2% hyclone, prepare to carry out transfection.
(3) prepare H1,5-FAM-Dbait solution according to N/P=12:1,5-FAM-Dbait consumption is 0.1nmol/2ml culture medium/hole.
(4), under lucifuge, vortex oscillation condition, by H1 solution instillation 5-FAM-Dbait solution, continue vortex and vibrate 15 seconds, mixing H1/Dbait transfection cocktail, room temperature lucifuge leaves standstill 20min.
(5) H1/Dbait transfection cocktail is slowly evenly added in 6 orifice plates, all around shake, make it fully mix, be placed in 37 DEG C, 5%CO
2continue in incubator to cultivate.
(6) cultivate after 24 hours, observe green fluorescence expression under inverted fluorescence microscope, determine transfection efficiency.
Embodiment 4.MTT method detects the toxicity of novel targeted nanoparticle to Prostatic cancer cell lines PC3 cell
(1) the PC3 cell being in exponential phase is seeded in 96 orifice plates, 5 × 10
3/ hole, with the RPMI1640 culture medium cellar culture containing 10% hyclone, 100 μ g/ml streptomycins, 100U/ml penicillin.After cell attachment, replace medium to antibiotic-free, RPMI1640 culture medium containing 0.05% hyclone, prepare to carry out transfection.
(2) experiment is divided into four groups: matched group, H1/GFP transfection group, Lip2000/GFP transfection group, positive controls (H
2o
2group).
(3) corresponding transfection process is after 24 hours, adds 20ulMTT solution (being diluted to 5ug/ul) in each hole, continues cultivation 4 hours in incubator.
(4) carefully abandon the culture medium in each hole in clean 96 orifice plates, every hole adds 200ulDMSO, and shaken at room temperature measures OD value after hatching 10min, and wavelength is 595nm.
(5) statistical analysis is carried out by each group of OD value importing GraphPadPrism5.0 software.
Embodiment 5. colony formation detects the impact of novel targeted nanoparticle on the clonality of Prostatic cancer cell lines PC3 cell
(1) the PC3 cell being in exponential phase is seeded in 6 orifice plates, 3 × 10
5/ hole, with the RPMI1640 culture medium cellar culture containing 10% hyclone, 100 μ g/ml streptomycins, 100U/ml penicillin.Be divided into 3 groups: matched group, H1/GFP group, H1/Dbait group.When cell fusion degree reaches 70%-80%, replace medium to antibiotic-free, RPMI1640 culture medium containing 2% hyclone, prepare to carry out transfection.
(2), after carrying out H1/Dbait and H1/GFP transfection according to above-mentioned transfection method, 37 DEG C are placed in, 5%CO
2continue in incubator to cultivate.
After (3) 4 ~ 6 hours, after matched group, H1/GFP group, H1/Dbait group 3 groups of cell dissociations, counting, be seeded in new six orifice plates according to proper density, continue to cultivate by the RPMI1640 culture medium containing 10% hyclone, 100 μ g/ml streptomycins, 100U/ml penicillin.
(4) after cell is completely adherent, carry out cell irradiation, exposure dose is respectively: 0Gy, 2Gy, 4Gy, 6Gy, 8Gy.Close rate is 300cGy/min.
(5) cultivation about 2 weeks is continued after irradiating, period periodic replacement culture medium (changing once for about 2 ~ 3 days).
(6) treat in culture dish, to occur macroscopic clone, stop cultivating, carry out violet staining.
I. abandon supernatant, carefully embathe 2 times with PBS.
II. add 4% paraformaldehyde and fix 15min.
III. abandon fixative, carefully embathe 2 times with PBS.
IV. add 1ml/ hole violet staining liquid, after dyeing 20 ~ 30min, slowly wash away dyeing liquor with flowing water, air natural is dry.
(7) take pictures, calculate cloning efficiency=(clone's number/inoculating cell number) × 100%.
(8) GraphPadPrism5.0 software matching one-hit multitarget model and Linear quadratic model curve is utilized.
Embodiment 6.WesternBlot method detects novel targeted nanoparticle to DNA damage label γ-H in Prostatic cancer cell lines PC3 cell
2the impact of AX expression
(1) the PC3 cell being in exponential phase is seeded in 6 orifice plates, 3 × 10
5/ hole, with the RPMI1640 culture medium cellar culture containing 10% hyclone, 100 μ g/ml streptomycins, 100U/ml penicillin.When cell fusion degree reaches 70%-80%, replace medium to antibiotic-free, RPMI1640 culture medium containing 2% hyclone, prepare to carry out transfection.
(2), after carrying out H1/Dbait and H1/GFP transfection according to above-mentioned transfection method, 37 DEG C are placed in, 5%CO
2continue in incubator to cultivate.
(3) transfection is after 4 ~ 6 hours, carries out cell irradiation, and exposure dose is 4Gy, and close rate is 300cGy/min.
(4) 37 DEG C, 5%CO
2continue cultivation in incubator after 24 hours, abandon clean old culture medium, after cleaning 2 times with the PBS of pre-cooling, exhaustion PBS, goes to WesternBlot.
Add 150ulRIPA lysate+1.5ul protease inhibitor+1.5ul protease inhibitor I+1.5ul inhibitors of phosphatases II in (5) the six each holes of orifice plate, cross rocks the even six orifice plate bottom surfaces of paving, cracking 30min on ice.
(6) collect, extract total protein of cell, 4 DEG C, after the centrifugal 10min of 12000 × g, supernatant is transferred in new EP pipe, abandons precipitation.
(7), after BCA method measures protein concentration, adjustment protein concentration is equal to each histone concentration.
(8) add appropriate 5 × SDS sample-loading buffer, 100 DEG C are boiled 10min and carry out albuminous degeneration.
(9) electrophoresis: concentrated gum concentration is 5%, resolving gel concentration is 10%.During electrophoresis, concentrated glue voltage is 80V, and separation gel voltage is 120V.When bromophenol blue is run to separation gel, stop electrophoresis.
(10) transferring film: constant voltage transferring film, voltage is 100V, and the time is 100min.
(11) close: under room temperature, 5% defatted milk powder (TBST buffer) closes 2 hours.
(12) primary antibodie is hatched: primary antibodie diluted concentration is 1:1000,4 DEG C of overnight incubation.
(13) next day, reclaim primary antibodie, wash film with TBST buffer, 5min/ time × 3 times.
(14) two anti-hatch: two anti-diluted concentrations are 1:10000, incubated at room 2 hours.
(15) film is washed with TBST buffer, 5min/ time × 5 times.
(16) Chemiluminescence Apparatus develops and preservation of taking pictures.
(17) with ImageJ software, gray analysis is carried out to result.
The novel targeted nanoparticle of embodiment 7. immuno-fluorescence assay is to DNA damage label γ-H in Prostatic cancer cell lines PC3 cell
2the impact of AX expression
(1) experiment grouping: matched group, H1/Dbait group, irradiation group merely, irradiation associating H1/Dbait group.
(2) coverslip is soaked in 75% ethanol spends the night, after secondary daily aseptic PBS rinses, put into 6 orifice plates.
(3) the PC3 cell of exponential phase is seeded to is covered with in 6 orifice plates of coverslip, 3 × 10
5/ hole, often kind of process factor arranges 3 multiple holes.With the RPMI1640 culture medium cellar culture containing 10% hyclone, 100 μ g/ml streptomycins, 100U/ml penicillin.
(4) when cell fusion degree reaches 70%-80%, replace medium to antibiotic-free, RPMI1640 culture medium containing 2%FBS, prepare to carry out transfection according to N/P=12:1.
(5) H1/Dbait transfection method is the same.
(6) irradiate after H1/Dbait transfection 4 ~ 6h, exposure dose is 4Gy, and close rate is 300cGy/min.
(7), after irradiating 24h, discard old culture medium, every hole adds 2mlPBS, rinsing 3min × 3 time.
(8) sucking-off PBS, every hole adds 4% paraformaldehyde 1ml of pre-cooling, and room temperature fixes 20min.
(9) sucking-off 4% paraformaldehyde, every hole adds 2mlPBS, rinsing 5min × 3 time.
(10) sucking-off PBS, every hole adds 1ml0.5%TritonX-100, and room temperature changes process 10min thoroughly.
(11) sucking-off 0.5%TritonX-100, every hole adds 2mlPBS, rinsing 5min × 3 time.
(12) sucking-off PBS, every hole adds 1ml2%BSA (preparing with PBS), closes 1h under room temperature.
(13) sucking-off 2%BSA, does not wash.
(14) every hole adds 50ul primary antibodie (using primary antibodie diluted to specifications) and covers coverslip, and 4 DEG C are spent the night.
(15) sucking-off next day primary antibodie, every hole adds 2mlPBS, rinsing 3min × 3 time.
(16) sucking-off PBS, every hole adds 50ulFITC-bis-anti-(diluting with 2%BSA to specifications) and covers coverslip, incubated at room 1h.
(17) sucking-off two resists, and every hole adds 2mlPBS, rinsing 5min × 3 time.
(18) every hole adds 4 Hochest33342, and jog mixes, incubated at room 20min.
(19) sucking-off PBS, every hole adds 2mlPBS, rinsing 5min × 3 time.
(20) take out coverslip, to take pictures under fluorescence microscope and to preserve just putting after mounting.
Embodiment 8.WesternBlot detects the impact of novel targeted nanoparticle on DNA damage response protein developed by molecule level in Prostatic cancer cell lines PC3 cell
Method is with embodiment 7, and the destination protein of detection is γ-H
2aX, DNA-PKcs, and be internal reference with GAPDH.
The novel targeted nanoparticle of embodiment 9. is on the impact of the nude mice of load transplanted tumor
(1) foundation of carcinoma of prostate nude mice model: will be in the Prostatic cancer cell lines PC3 cell seeding of logarithmic (log) phase growth in nude mice right lower extremity, cell quantity: 5,000,000/100ul/ only.
(2) treat that gross tumor volume grows to 100mm
3left and right, different according to intervening measure, divide into groups: matched group (Control), irradiation group merely (IR), irradiation associating H1/GFP group (IR+H1/GFP), irradiation associating H1/Dbait group (IR+H1/Dbait).Often organize 9 tumor bearing nude mices.
(3) nude mice intervenes process: matched group: intratumor injection 5% glucose; Simple irradiation group: 3Gy/ times/day × 3 days (Continuous irradiation 3 days); Irradiate associating H1/GFP group: 3Gy/ times/day × 3 days (Continuous irradiation 3 days), each pre-irradiation carries out H1/GFP mixed liquor intratumor injection for 5 hours; Irradiate associating H1/Dbait group: 3Gy/ times/day × 3 days (Continuous irradiation 3 days), each pre-irradiation carries out H1/Dbait mixed liquor intratumor injection for 5 hours.
Intervene process (4) 3 times and terminate latter 24 hours, often organize random execution 3 nude mices, taking-up tumor tissues carries out westernblot and analyzes and SABC detection.
(5) all the other nude mices are for measuring gross tumor volume and observing nude mice life span.Gross tumor volume measuring method is: within every 3 days, measure once, each repeated measure 3 times, averages.Gross tumor volume computing formula is: gross tumor volume=length × wide × wide/2.
(6) related data is imported in GraphPadPrism5.0 software carry out statistical analysis, and draw tumor volume growth curve and nude mice survival curve.
Claims (7)
1. a novel targeted nanoparticle, is characterized in that: described nanoparticle is made up of the double chain DNA molecule of folate-targeted, biodegradable positron polymer support PEI-CyD-FA (H1) load small fragment hair clip spline structure.
2. novel targeted nanoparticle according to claim 1, is characterized in that: the double chain DNA molecule <50bp of described small fragment hair clip spline structure.
3. novel targeted nanoparticle according to claim 1, is characterized in that: the double chain DNA molecule of described small fragment hair clip spline structure is Novel radioactive sensitizer Dbait molecule, and its size is 32bp.
4. novel targeted nanoparticle according to claim 1, is characterized in that: the particle diameter of described nanoparticle is 100 ~ 200 nanometers, and Zeta potential is 10mV ~ 20mV.
5. the preparation method of novel targeted nanoparticle according to claim 1, is characterized in that: comprise the following steps:
(1) get positron polymer support PEI-CyD-FA (H1) to be dissolved in sterilizing distilled water, obtain solution 1, room temperature, lucifuge leave standstill 5 minutes;
(2) the double chain DNA molecule Dbait getting small fragment hair clip spline structure is dissolved in sterilizing distilled water, obtains solution 2, and room temperature leaves standstill 5 minutes;
(3) instill in solution 2 by solution 1 under lucifuge, vortex oscillation, continue vortex oscillation 15 seconds, solution 1 and solution 2 are fully mixed, and lucifuge, room temperature leave standstill and obtain novel targeted nanoparticle H1/Dbait after 20 minutes, namely can be used for follow-up related experiment.
6. novel targeted nanoparticle according to claim 1 is used for the purposes of tumour radiotherapy.
7. the purposes of novel targeted nanoparticle according to claim 6, is characterized in that: described tumor is carcinoma of prostate or colorectal cancer or hepatocarcinoma or pulmonary carcinoma or melanoma or breast carcinoma or ovarian cancer or cancer of pancreas or the esophageal carcinoma or gastric cancer or uterus carcinoma or laryngeal carcinoma or thyroid carcinoma.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114099639A (en) * | 2021-11-25 | 2022-03-01 | 徐州医科大学 | H1-pHSP65 nano vaccine, preparation method and application thereof |
| AU2019370892B2 (en) * | 2018-11-01 | 2023-04-27 | Alpha Tau Medical Ltd. | Intratumoral alpha-emitter radiation and activation of cytoplasmatic sensors for intracellular pathogen |
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2015
- 2015-08-25 CN CN201510528034.2A patent/CN105031648A/en active Pending
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| JULIAN BIAU等: ""A Preclinical Study Combining the DNA Repair Inhibitor Dbait with Radiotherapy for the Treatment of melanoma"", 《NEOPLASIA》 * |
| N BERTHAULT等: ""Comparison of distribution and activity of nanoparticles with short interfering DNA(Dbait) in various living systems"", 《CANCER GENE THERAPY》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2019370892B2 (en) * | 2018-11-01 | 2023-04-27 | Alpha Tau Medical Ltd. | Intratumoral alpha-emitter radiation and activation of cytoplasmatic sensors for intracellular pathogen |
| CN114099639A (en) * | 2021-11-25 | 2022-03-01 | 徐州医科大学 | H1-pHSP65 nano vaccine, preparation method and application thereof |
| CN114099639B (en) * | 2021-11-25 | 2024-03-01 | 徐州医科大学 | H1-pHSP65 nanometer vaccine, preparation method and application thereof |
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