CN102614524A - Brain tumour targeting gene delivery composite modified by low density lipoprotein receptor associated protein ligand polypeptide - Google Patents
Brain tumour targeting gene delivery composite modified by low density lipoprotein receptor associated protein ligand polypeptide Download PDFInfo
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Abstract
The invention, belonging to the field of biotechnology, relates to a brain tumor targeting gene delivery composite modified by low density lipoprotein receptor associated protein ligand polypeptide. The brain tumor targeting gene delivery composite has a dual targeting effect of BBB and glioma. Therapeutic genes can express and kill tumor cells in glioma cells, and can release gene product TRAIL protein into the environment outside the cells to combine with tumor cells by the means of cell paracrine after expressing in BCECs and then have a killing effect, thus aiming at the infiltrative growth characteristic of glioma in the brain, the brain tumor targeting gene delivery composite can kill the main glioma and simultaneously effectively inhibit the diffusion of scattered tumor cells.
Description
Technical field
The invention belongs to biological technical field, relate to cerebral tumor target gene and pass and release complex, be specifically related to cerebral tumor target gene that the LDH receptor related protein ligand polypeptide modifies and pass and release complex.
Background technology
Research shows that blood brain barrier (being called for short BBB) can effectively stop exogenous harmful substance to get in the brain, thereby guarantees the stable of brain environment and brain function.BBB mainly is made up of brain capillary endothelial cell (being called for short BCECs), and intercellular tight connection stops most drug, and for example antibiotic, antitumor drug, neuropeptide and other active medicines get into the nervus centralis complex.Research shows, nearly all macromolecular drug such as genomic medicine and surpass 98% small-molecule drug and can't see through BBB.
At present, increase strategy that medicine transmits in brain and comprise two types of invasive (for example directly intracerebral injection) and Noninvasives.The Noninvasive strategy comprises that being prepared into prodrug, drug molecule directly links to each other with lipid components or targeting part, but these methods can make drug molecule lose the part curative effect.Nanometer administration complex is a kind of interior delivery strategies of non-invasive drug brain with wide application prospect.For genomic medicine, cation high molecular polymer non-virus carrier effectively condensation DNA forms nanoparticle, protects contained gene to avoid the DNA enzymatic degradation; Through modify molecule in nanometer administration composite surface with targeting property, for example monoclonal antibody, peptide class, agglutinin, saccharide, hormones and low-molecular-weight chemical compound etc. can obviously increase its accumulating at target site.
Be applied to the brain targeted drug based on the drug delivery complex of receptor-mediated mechanism and passed the field of releasing.Specific receptor on the known BBB of being present in has TfR, Insulin receptor INSR, IGF-1, EGF-R ELISA etc. at present.Existing report shows that LDH receptor related protein (being called for short LRP) can mediate its part and cross over the endotheliocyte on the BBB.The peptide class Angiopeps of family of previous report is the Ku Erci territory that derives from aprotinin and LDH receptor related protein.Wherein Angiopep-2 (TFFYGGSRGKRNNFKTEEY, molecular weight 2.4 kD) can go up the LRP mediation of expressing through BBB to get into one section small peptide of being made up of 19 amino acid residues in the brain.Research shows, the brain efficient of going into of Angiopep-2 is 70 times of classical brain targeting head base transferrins.And LRP is all expressed on BCECs and glioma cell surface, therefore selects for use Angiopep-2 decorated nanometer administration complex might realize brain targeting or the effect of BBB-glioma dual-target.
Summary of the invention
The present invention seeks to go into problems such as brain efficient is low, toxic and side effects is big, provide new cerebral tumor target gene to pass and release complex to the pharmaceutical preparation of clinical existing treatment nervus centralis complex disease.Being specifically related to cerebral tumor target gene that the LDH receptor related protein ligand polypeptide modifies passs and releases complex.
The present invention adopts Angiopep-2 as brain targeting part; Use the cation high molecular polymer to be carrier is carrier; Connect Angiopep-2 through hydrophilic polymer and make up gene and pass and release carrier, the gene that is obtained is passed and is released carrier and plasmid DNA construction and form the big or small gene of 50～300 nanometers and pass that to release complex be that cerebral tumor target gene of the present invention is passed and released complex.
Particularly; The cerebral tumor target gene that LDH receptor related protein ligand polypeptide of the present invention is modified is passed and is released complex; It is characterized in that; Macromolecule carrier and the DNA modified by Angiopep-2 constitute, and the macromolecule carrier that described Angiopep-2 modifies is made up of cation high molecular material, hydrophilic polymer and Angiopep-2; The mass ratio of described cation high molecular material and DNA is 2 ~ 40:1.
Cation high molecular material-hydrophilic polymer-Angiopep-2/DNA gene that the present invention makes up is passed and is released complex; Can obviously improve transfection efficiency and the expression of therapeutic gene, especially the transfection efficiency of cerebral glioma therapeutic gene and expression at brain.
Among the present invention, described cation high molecular material is selected from cation dendrimer polyamide-amide (being called for short PAMAM) and dendroid polylysine.
Among the present invention, described cerebral glioma therapeutic gene is the codes for tumor necrosin apoptosis induction ligand gene of (being called for short TRAIL).
Among the present invention, the molecular proportion of hydrophilic polymer and cation high molecular material is 2 ~ 20:1, and the molecular proportion of Angiopep-2 and cation high molecular material is 1 ~ 10:1.
The invention provides cerebral tumor target gene and pass the method for preparing of releasing complex, it is characterized in that, adopt Angiopep-2 as targeting head base, the cation high molecular material is a carrier is carrier, connects Angiopep-2 through hydrophilic polymer and constructs macromolecule carrier; Macromolecule carrier that is obtained and DNA form the complex of 50～300 nanometers size, and the cation high molecular material wherein and the mass ratio of DNA are 2 ~ 40:1.
Macromolecule carrier of the present invention, wherein cation high molecular material, hydrophilic polymer and Angiopep-2 are connected with covalent manner.
Described macromolecule carrier prepares through following step:
Cation high molecular material and hydrophilic polymer are dissolved in pH value 7.8～8.2 phosphate buffers with the molecular proportion of 1:2 ~ 20; Stirring at room reaction 1～3 hour; The group generation specific reaction that wherein contains; Generate cation high molecular material-hydrophilic polymer, ultrafiltration is removed unreacted hydrophilic polymer and is replaced by pH value 6.8～7.2 phosphate buffers; Simultaneously; Sulfydryl is introduced in Angiopep-2 and sulfhydrylization reagent reaction back; Generate cation high molecular material-hydrophilic polymer-lactoferrin after 12～36 hours with the molecular proportion of 1～10:1 and the special group stirring at room reaction on cation high molecular material-hydrophilic polymer, molecular exclusion chromatography is removed unreacted Angiopep-2.
The invention provides described cerebral tumor target gene and pass the method for preparing of releasing complex:
PAMAM and the PEG that contains bifunctional group are dissolved in the phosphate buffer (being called for short PBS) of pH 8.0; Stirring at room reaction 2 hours; Generate PAMAM-PEG; Unreacted PEG is removed in ultrafiltration and exchange buffering liquid is the PBS of pH 7.0, adds the Angiopep solution of sulfhydrylation, and the stirring at room reaction generates cerebral tumor target gene and passs and release complex (PAMAM-PEG-Angiopep) after 24 hours.Wherein the molecular proportion of PAMAM and PEG is 1:2 ~ 20, and the molecular proportion of PAMAM and Angiopep is 1:1 ~ 10.
The present invention carries out structure through nuclear magnetic resonance technique and identifies.Weighing PAMAM-PEG-Angiopep 1.0 mg are dissolved in the 0.5 ml heavy water, and Mercury Plus 400 MHz NMR spectrometer with superconducting magnet are identified the structure of macromolecule carrier.Simultaneously, bifunctional group PEG 1.0 mg are dissolved in the 0.5 ml heavy water, Mercury Plus 400 MHz NMR spectrometer with superconducting magnet are identified structure, analyze the two collection of illustrative plates, and the result confirms, successfully synthetic PAMAM-PEG-Angiopep.
Above-mentioned synthetic PAMAM-PEG-Angiopep macromolecule carrier is dissolved in the solution that is made into variable concentrations among the PBS of an amount of pH 7.4; The reporter gene DNA is dissolved in the solution that is mixed with 100 μ g/ml in the 50 an amount of mM metabisulfite solutions, and both equal-volume whirlpool 30 s mixings are processed the PAMAM-PEG-Angiopep/DNA gene and are passed and release complex under the room temperature.Wherein the mass ratio of PAMAM-PEG-Angiopep macromolecule carrier and DNA is 2 ~ 40:1 (in the quality of PAMAM).
Complex is released in passing through fluorescently-labeled PAMAM-PEG-Angiopep/DNA gene of above-mentioned preparation, through the nude mice tail vein injection, places Maestro with the dosage of DNA/ mice of 50 μ g in 2 hours
TMObserve in the 2 living imaging appearance, the result shows, and without the PAMAM/DNA nanoparticle of modifying relatively, the PAMAM-PEG-Angiopep/DNA gene of modifying through Angiopep is passed and released complex and can obviously improve the picked-up of gene at brain.
Complex is released in passing through radiolabeled PAMAM-PEG-Angiopep/DNA gene of above-mentioned preparation; With the dosage of DNA/ mice of 50 μ g through the Balb/c mouse tail vein injection; Getting brain, the heart, liver, spleen, lung, kidney after 2 hours detects; The result shows, and without the PAMAM/DNA nanoparticle of modifying relatively, the PAMAM-PEG-Angiopep/DNA gene of modifying through Angiopep is passed and released complex and can improve the picked-up of gene at brain.
Complex is released in passing through fluorescently-labeled PAMAM-PEG-Angiopep/DNA gene of the above-mentioned preparation of lotus cerebral glioma nude mice tail vein injection, and dosage is DNA/ mices of 50 μ g, places Maestro in 2 hours
TMObserve in the 2 living imaging appearance; Take out the observation of exsomatizing of internal organs such as brain, the heart, liver, spleen, lung, kidney subsequently; The result shows; With without the PAMAM/DNA nanoparticle of modifying relatively, the PAMAM-PEG-Angiopep/DNA gene of modifying through Angiopep is passed and is released complex and can improve the picked-up of gene at brain.
Lotus cerebral glioma ICR mice was set up the back the 8th day at tumor model; The 10th day, the 12nd day respectively the PAMAM-PEG-Angiopep/DNA gene of the above-mentioned preparation of tail vein injection pass and release complex, each dosage is DNA/ mices of 50 μ g; The record mice time-to-live; The result shows, and without the PAMAM/DNA nanoparticle of modifying relatively, the PAMAM-PEG-Angiopep/DNA gene of modifying through Angiopep is passed and released the time-to-live that complex can effectively improve tumor-bearing mice.
Lotus cerebral glioma ICR mice was set up the back the 8th day at tumor model, the 10th day, the 12nd day respectively the PAMAM-PEG-Angiopep/DNA gene of the above-mentioned preparation of tail vein injection pass and release complex, each dosage is DNA/ mices of 50 μ g, gets full brain, 4 on the 15th day
oIn C, 4% the paraformaldehyde solution fixing 48 hours, 4
oDehydration is 6 hours in C, 15% the sucrose solution, and 4
oDehydration is 24 hours in C, 30% the sucrose solution; Embedding, carry out frozen section after freezing; Thickness is 20 μ m, carries out TUNEL original position apoptosis subsequently and detects, and the result shows; With without the PAMAM/DNA nanoparticle of modifying relatively, the PAMAM-PEG-Angiopep/DNA gene of modifying through Angiopep is passed and is released complex and can more effectively induce glioma cell generation apoptosis.
Outstanding advantage of the present invention is; Employing has brain targeting head base---the Angiopep-2 of clinical practice potentiality; Modification cerebral tumor target gene is passed and is released complex; Can increase of the picked-up of cerebral glioma therapeutic gene, particularly through significantly improving the accumulation of genomic medicine after the administration of AT intravenous injection mode at brain at BCECs.Compare with existing commercial preparation such as oral temozolomide, brain targeting efficient significantly improves.
The present invention makes up cerebral tumor target gene and passs and release targeting head base Angiopep-2 and its receptor LRP that complex adopts and have high-affinity; And LRP on the BCECs surface and the glioma cell surface expression is all arranged; Therefore this gene is passed and is released complex and have BBB and the effect of glioma dual-target; Tumor cell can expressed and kill to therapeutic gene in glioma cell; Also can in BCECs, express the mode of back through the cell paracrine and the gene outcome trail protein is released into the extracellular combines, play lethal effect with tumor cell.To the infiltrative growth characteristics of cerebral glioma in brain, this nanometer administration complex expection can effectively suppress the diffusion of fragmentary glioma cell when killing the glioma main body.
Description of drawings
Fig. 1,Nuclear magnetic resonance map,
Wherein, A is PEG,
B is PAMAM-PEG-Angiopep.
Fig. 2,The picked-up of PAMAM-PEG-Angiopep on BCECs,
Wherein, A-D is the fluorescence microscope qualitative observation,
E-H is the flow cytometer quantitative assay,
A, the concentration of PAMAM-PEG-Angiopep is 0.035 μ M among the B,
C, the concentration of PAMAM-PEG-Angiopep is 0.105 μ M among the D,
E, the concentration of PAMAM-PEG-Angiopep is 0. 35 μ M among the F,
G, the concentration of PAMAM-PEG-Angiopep is 0.105 μ M among the H.
Fig. 3,The picked-up of macromolecule carrier on BCECs,
Wherein, A is PAMAM, 37
0C,
B is PAMAM-PEG-Angiopep, 37
0C,
C is PAMAM-PEG-Angiopep, 4
0C,
D is PAMAM-PEG-Angiopep+Angiopep-2,37
0C,
E is PAMAM-PEG-Angiopep+RAP, 37
0C,
F is PAMAM-PEG-Angiopep+lactoferrin, 37
0C.
Fig. 4,Gene is passed and is released the picked-up fluorescence microscope of complex on BCECs,
Wherein, A is PAMAM/DNA-Angiopep/DNA,
B is PAMAM/DNA,
C is PAMAM-PEG-Angiopep/DNA+poly-D-lysine,
D is PAMAM-PEG-Angiopep/DNA+phenylarsenic oxide,
E is PAMAM-PEG-Angiopep/DNA+Fei Liping,
F is PAMAM-PEG-Angiopep/DNA+colchicine,
G is PAMAM-PEG-Angiopep/DNA+Angiopep-2,
H is PAMAM-PEG-Angiopep/DNA+RAP,
I is PAMAM-PEG-Angiopep/DNA+lactoferrin.
Fig. 5,The picked-up Laser Scanning Confocal Microscope of PAMAM-PEG-Angiopep/DNA on BCECs observed,
Wherein, A-D is 15 minutes,
B-E is 60 minutes.
Fig. 6, gene is passed and is released complex and stride BCECs monofilm transhipment.
Fig. 7, gene is passed and is released distribution living imaging observation in the complex nude mouse,
Wherein, A is PAMAM/DNA,
B is PAMAM-PEG-Angiopep/DNA.
Fig. 8, gene is passed and is released distribution radioactive label mensuration in the complex mice body,
Wherein, A distributes in the brain,
B is that other internal organs (heart, liver, spleen, lung, kidney) distribute.
Fig. 9,Frozen section investigation different genes is passed and is released the expression of complex at cerebral tissue,
Wherein, A-D passs the expression of results of releasing complex for the PAMAM/DNA gene,
E-H passs the expression of results of releasing complex for the PAMAM-PEG-Angiopep/DNA gene,
A, E: cerebral cortex frozen section figure,
B, F: striatum frozen section figure,
C, G: Hippocampus frozen section figure,
D, H: black substance frozen section figure.
Figure 10, gene is passed and is released complex distribution living imaging observation in the body of lotus cerebral glioma nude mice,
Wherein, A is the nude mice living imaging,
B is the brain fluorescence imaging that exsomatizes,
C is internal organs (brain, the heart, liver, spleen, lung, the kidney) fluorescence imaging that exsomatizes.
Figure 11, lotus cerebral glioma mice survival curve.
Figure 12, original position TUNEL detects glioma apoptosis in the lotus cerebral glioma mouse brain,
Wherein, A is a normal saline,
B is PAMAM-PEG-Angiopep/pGL2,
C is temozolomide's capsule,
D is free pORF-TRAIL plasmid,
E is PAMAM/pORF-TRAIL,
F is PAMAM-PEG-Angiopep/pORF-TRAIL,
Yellow line indication glioma edge, red arrow indication apoptosis zone.
The specific embodiment
PAMAM and the PEG that contains bifunctional group are dissolved in according to molecular proportion 1:2 among the PBS of pH 8.0, and stirring at room reaction 2 hours generates PAMAM-PEG, and unreacted PEG is removed in ultrafiltration and exchange buffering liquid is the PBS of pH 7.0.Angiopep mixes according to the 1:1 molecular proportion with PAMAM-PEG, and the stirring at room reaction generated PAMAM-PEG-Angiopep after 24 hours.
PAMAM and the PEG that contains bifunctional group are dissolved in according to molecular proportion 1:5 among the PBS of pH 8.0, and stirring at room reaction 2 hours generates PAMAM-PEG, and unreacted PEG is removed in ultrafiltration and exchange buffering liquid is the PBS of pH 7.0.Angiopep mixes according to the 1:1 molecular proportion with PAMAM-PEG, and the stirring at room reaction generated PAMAM-PEG-Angiopep after 24 hours.
Embodiment 3
PAMAM-PEG-Angiopep macromolecule carrier 1.0 mg of embodiment 1 preparation are dissolved in the 0.5 ml heavy water, and Mercury Plus 400 MHz NMR spectrometer with superconducting magnet are identified the structure of macromolecule carrier.Simultaneously, bifunctional group PEG 1.0 mg are dissolved in the 0.5 ml heavy water, Mercury Plus 400 MHz NMR spectrometer with superconducting magnet are identified structure.Analyze the two collection of illustrative plates, PAMAM-PEG-Angiopep (as shown in Figure 1) is synthesized in the success of reaching a conclusion.
Embodiment 4
Quantitatively take by weighing BODIPY, be dissolved in 100 mM NaHCO
3Be mixed with 200 μ g/ml storing solutions in the solution, keep in Dark Place, use within 3 days.According to the ratio of BODIPY:PAMAM 10:1 (mol/mol), 4
oC lucifuge stirring reaction 12 h make BODIPY-PAMAM.BODIPY-PAMAM and the PEG that contains bifunctional group are dissolved in according to molecular proportion 1:2 among the PBS of pH 8.0, and stirring at room reaction 2 hours generates BODIPY-PAMAM-PEG, and unreacted PEG is removed in ultrafiltration and exchange buffering liquid is the PBS of pH 7.0.Angiopep mixes according to the 1:1 molecular proportion with BODIPY-PAMAM-PEG, and the stirring at room reaction generated BODIPY-PAMAM-PEG-Angiopep after 24 hours.
Using the PBS of pH7.4 to be diluted to PAMAM concentration the BODIPY-PAMAM-PEG-Angiopep of embodiment 4 preparation is 0.035 μ M, 0.105 μ M, 0.35 μ M and 1.05 μ M, with the BCECs cell in 37
oC, 5%CO
2Hatched in the incubator 30 minutes; Adopt the PBS solution rinse cell of pH 7.4 to adopt OLYMPUS IX 71 microscopic examinations picked-up result and take pictures (200 times of amplifications); Collect a part of cell in addition and carry out quantitative assay in FACSCalibur system flow cytometer; The result shows that the cellular uptake of BODIPY-PAMAM-PEG-Angiopep increases (as shown in Figure 2) along with the concentration increase.
Embodiment 6
BODIPY-PAMAM that embodiment 4 is made and BODIPY-PAMAM-PEG-Angiopep and BCECs are in 37
oC, 5%CO
2Hatched in the incubator 30 minutes; BODIPY-PAMAM-PEG-Angiopep that embodiment 4 is made and BCECs are in 4
oC, 5%CO
2Hatched in the incubator 30 minutes; Adopt the PBS solution rinse cell of pH 7.4; Adopt OLYMPUS IX 71 microscopic examinations picked-up result and take pictures (200 times of amplifications); The result shows that constructed PAMAM-PEG-Angiopep macromolecule carrier has obvious increase than the cellular uptake efficient of PAMAM, when low temperature, then absorbs minimizing (as shown in Figure 3).
Embodiment 7
Using the PBS configuration concentration of pH 7.4 is the Angiopep-2 solution of 100 μ g/mL; The PBS configuration concentration that uses pH 7.4 is RAP (the being called for short RAP) solution of 20 nM; The PBS configuration concentration that uses pH 7.4 is lactoferrin (the being called for short Lf) solution of 1 mg/mL.
Embodiment 8
Using the PBS configuration concentration of pH 7.4 is the Angiopep-2 solution of 200 μ g/mL; Using the PBS configuration concentration of pH 7.4 is the RAP solution of 40 nM; Using the PBS configuration concentration of pH 7.4 is the Lf solution of 2 mg/mL.
Embodiment 9
With the various solution of embodiment 7 preparation respectively with BCECs in 37
oC, 5%CO
2Hatched in the incubator 10 minutes, and adopted the PBS solution rinse cell of pH 7.4; Then with the PAMAM-PEG-Angiopep solution of embodiment 1 preparation respectively with after the various solution equal-volumes of embodiment 8 preparations mix, respectively with BCECs in 37
oC, 5%CO
2Hatched in the incubator 30 minutes; Adopt the PBS solution rinse cell of pH 7.4; Adopt OLYMPUS IX 71 microscopic examinations picked-up result and take pictures (200 times of amplifications); The result shows that Angiopep-2, RAP, Lf all can reduce the picked-up (as shown in Figure 3) of constructed PAMAM-PEG-Angiopep macromolecule carrier on BCECs.
The PAMAM-PEG-Angiopep macromolecule carrier of embodiment 1 preparation is dissolved in the solution that is made into 1 mg/ml (with the Mass Calculation of PAMAM) among the PBS of an amount of pH 7.4; PEGFP-N2 GFP egfp grain DNA is dissolved in the solution that is mixed with 100 μ g/ml in the 50 an amount of mM metabisulfite solutions, and both equal-volume whirlpool 30 s mixings are processed the PAMAM-PEG-Angiopep/DNA gene and are passed and release complex under the room temperature.
Embodiment 11
The PAMAM-PEG-Angiopep macromolecule carrier of embodiment 1 preparation is dissolved in the solution that is made into 1 mg/ml (with the Mass Calculation of PAMAM) among the PBS of an amount of pH 7.4; PGL2-Control Vector luciferase plasmids DNA is dissolved in the solution that is mixed with 100 μ g/ml in the 50 an amount of mM metabisulfite solutions, and both equal-volume whirlpool 30 s mixings are processed PAMAM-PEG-Angiopep/DNA gene and are passed and release complex under the room temperature.
Embodiment 12
The PAMAM-PEG-Angiopep macromolecule carrier of embodiment 1 preparation is dissolved in the solution that is made into 1 mg/ml (with the Mass Calculation of PAMAM) among the PBS of an amount of pH 7.4; The pORF-TRAIL DNA is dissolved in the solution that is mixed with 100 μ g/ml in the 50 an amount of mM metabisulfite solutions, and both equal-volume whirlpool 30 s mixings are processed PAMAM-PEG-Angiopep/DNA gene and are passed and release complex under the room temperature.
Embodiment 13
Using the PBS configuration concentration of pH 7.4 is the phenylarsenic oxide solution of 2.5 μ M; Using the PBS configuration concentration of pH 7.4 is the Fei Liping solution of 0.5 μ g/mL; Using the PBS configuration concentration of pH 7.4 is 2.5 μ M colchicine solutions; Using the PBS configuration concentration of pH 7.4 is the L-Poly-L-Lysine Solution of 25 μ g/mL.
Embodiment 14
Using the PBS configuration concentration of pH 7.4 is the phenylarsenic oxide solution of 5 μ M; Using the PBS configuration concentration of pH 7.4 is the Fei Liping solution of 1 μ g/mL; Using the PBS configuration concentration of pH 7.4 is 5 μ M colchicine solutions; Using the PBS configuration concentration of pH 7.4 is the L-Poly-L-Lysine Solution of 50 μ g/mL.
With the various solution of embodiment 7 and embodiment 13 preparations respectively with BCECs in 37
oC, 5%CO
2Hatched in the incubator 10 minutes, and adopted the PBS solution rinse cell of pH 7.4; Then with the PAMAM-PEG-Angiopep/DNA solution of embodiment 11 preparation respectively with after the various solution equal-volumes of embodiment 8 and embodiment 13 preparations mix, respectively with BCECs in 37
oC, 5%CO
2Hatched in the incubator 30 minutes; Adopt the PBS solution rinse cell of pH 7.4; Adopt OLYMPUS IX 71 microscopic examinations picked-up result and take pictures (200 times of amplifications); The result shows, Angiopep-2, RAP, Lf, phenylarsenic oxide, Fei Liping, colchicine, L-poly-D-lysine all can reduce PAMAM-PEG-Angiopep/DNA gene that the present invention makes up to be passed and release the picked-up of complex on BCECs, sees accompanying drawing 4.
Embodiment 16
With the PAMAM-PEG-Angiopep/DNA solution of embodiment 11 preparation and BCECs in 37
oC, 5%CO
2Hatched in the incubator 15 minutes; Incubation time finishes preceding 10 minutes adding fluorescent dye Lysotracker Green acid organelle is dyeed; Adopt the PBS solution rinse cell of pH 7.4; Adopt TCS SP2 AOBS Laser Scanning Confocal Microscope to observe gene and pass the picked-up situation of releasing complex, see accompanying drawing 5.
Embodiment 17
With the PAMAM-PEG-Angiopep/DNA solution of embodiment 11 preparation and BCECs in 37
oC, 5%CO
2Hatched in the incubator 60 minutes; Incubation time finishes preceding 10 minutes adding fluorescent dye Lysotracker Green acid organelle is dyeed; Adopt the PBS solution rinse cell of pH 7.4; Adopt TCS SP2 AOBS Laser Scanning Confocal Microscope to observe gene and pass the picked-up situation of releasing complex, see accompanying drawing 5.
Embodiment 18
The PAMAM-PEG-Angiopep/DNA gene of nude mice tail vein injection embodiment 11 preparation is passed and is released complex, and dosage is DNA/ mices of 50 μ g, places Maestro in 2 hours
TMObserve in the 2 living imaging appearance, the result shows, the gene of the present invention's preparation is passed and released complex and can improve the picked-up of gene at brain, sees Fig. 7.
Embodiment 19
The PAMAM-PEG-Angiopep/DNA gene of Balb/c mouse tail vein injection embodiment 11 preparations is passed and is released complex; Dosage is DNA/ mices of 50 μ g; Getting brain, the heart, liver, spleen, lung, kidney after 2 hours detects; The result shows, the gene of the present invention's preparation is passed and released complex and can improve the picked-up of gene at brain, sees Fig. 8.
The PAMAM-PEG-Angiopep/DNA gene of Balb/c mouse tail vein injection embodiment 10 preparations is passed and is released complex, and dosage is DNA/ mices of 50 μ g, gets full brain, 4 after 2 days
oIn C, 4% the paraformaldehyde solution fixing 48 hours, 4
oDehydration is 24 hours in C, 15% the sucrose solution, and 4
oDehydration is 24 hours in C, 30% the sucrose solution; Embedding, carry out frozen section after freezing, thickness is 20 μ m, adopts fluorometric reagent DAPI (300 nM) to carry out nuclear staining; With the expression of results of green fluorescent protein in OLYMPUS IX 71 microscopic examination mouse brain cortexes, striatum, Hippocampus and the black substance and take pictures; The result shows, the gene of the present invention's preparation is passed and released complex brain targeting effect and significantly improve, and sees Fig. 9.
Embodiment 21
The C6 cell is through trypsinization, it is suspended in Hank ' the s liquid after centrifugal, and counting is regulated concentration to 5 * 10
7/ ml.Nude mice or ICR mouse peritoneal injection 5ml/kg 10% chloral hydrate anesthesia cut off the head hair, the long incision of scalp of vertical 1 cm of endocanthion line and head saggital midline intersection, and passivity is separated the exposure skull.In the other 1.8 mm place sphenotresias of opening of bregma.10 μ l microsyringes suck the above-mentioned cell suspension of 2 μ l, are fixed on the stereotaxic instrument, along dark 4 mm of the vertical inserting needle of boring; The withdraw of the needle 1 mm is (apart from cerebral dura mater; Be big brain striatum district), slowly inject C6 cell suspension, behind pin 5 min, slowly pull out pin; The art open country is cleaned with normal saline, and otch is sewed up knotting with sutures.
Embodiment 22
The PAMAM-PEG-Angiopep/DNA gene of lotus cerebral glioma nude mice tail vein injection embodiment 11 preparation is passed and is released complex, and dosage is DNA/ mices of 50 μ g, places Maestro in 2 hours
TMObserve in the 2 living imaging appearance, take out the observation of exsomatizing of internal organs such as brain, the heart, liver, spleen, lung, kidney subsequently, the result shows, the gene of the present invention's preparation is passed and released complex and can improve the picked-up of gene at brain, sees Figure 10.
Embodiment 23
Lotus cerebral glioma ICR mice was set up the back the 8th day at tumor model; The 10th day, the 12nd day respectively the PAMAM-PEG-Angiopep/DNA gene of tail vein injection embodiment 12 preparation pass and release complex, each dosage is DNA/ mices of 50 μ g; The record mice time-to-live; The result shows that the gene of the present invention's preparation is passed and released the time-to-live that complex can effectively improve tumor-bearing mice, sees Figure 11.
Embodiment 24
Lotus cerebral glioma ICR mice was set up the back the 8th day at tumor model, the 10th day, the 12nd day respectively the PAMAM-PEG-Angiopep/DNA gene of tail vein injection embodiment 12 preparations pass and release complex, each dosage is DNA/ mices of 50 μ g, gets full brain, 4 on the 15th day
oIn C, 4% the paraformaldehyde solution fixing 48 hours, 4
oDehydration is 6 hours in C, 15% the sucrose solution, and 4
oDehydration is 24 hours in C, 30% the sucrose solution; Embedding, carry out frozen section after freezing; Thickness is 20 μ m, carries out TUNEL original position apoptosis subsequently and detects, and the result shows; The PAMAM-PEG-Angiopep/DNA gene of the present invention preparation is passed and is released complex and can effectively induce glioma cell generation apoptosis, sees Figure 12.
The dendritic macromole PAMAM that adopts in the above-mentioned experiment of the present invention is available from Dendritech company, and PEG is available from the Jiankai Science and Technology Co., Ltd., Beijing, and polypeptide A ngiopep-2 is synthetic by Zhongtai Bio-Chem. Co., Ltd., Hangzhou.
Claims (7)
1. the cerebral tumor target gene of LDH receptor related protein ligand polypeptide modification is passed and is released complex; It is characterized in that; Macromolecule carrier and the DNA modified by Angiopep-2 constitute, and the macromolecule carrier that described Angiopep-2 modifies is made up of cation high molecular material, hydrophilic polymer and Angiopep-2; The mass ratio of described cation high molecular material and DNA is 2 ~ 40:1.
2. the cerebral tumor target gene of modifying by the described LDH receptor related protein ligand polypeptide of claim 1 is passed and is released complex; It is characterized in that; The macromolecule carrier that described Angiopep-2 modifies; Wherein the molecular proportion of hydrophilic polymer and cation high molecular material is 2 ~ 20:1, and the molecular proportion of Angiopep-2 and cation high molecular material is 1 ~ 10:1.
3. the cerebral tumor target gene of modifying by the described LDH receptor related protein ligand polypeptide of claim 1 is passed and is released complex; It is characterized in that described cation high molecular material is selected from cation dendrimer polyamide-amide or dendroid polylysine.
4. the cerebral tumor target gene of modifying by the described LDH receptor related protein ligand polypeptide of claim 1 is passed and is released complex; It is characterized in that; Described DNA is the cerebral glioma therapeutic gene, and this gene is the gene of codes for tumor necrosin apoptosis induction ligand.
5. the cerebral tumor target gene of the described LDH receptor related protein ligand polypeptide modification of claim 1 is passed the method for preparing of releasing complex; It is characterized in that; It comprises: adopt Angiopep-2 as targeting head base; The cation high molecular material is a carrier is carrier, connects Angiopep-2 through hydrophilic polymer and constructs macromolecule carrier; Macromolecule carrier that is obtained and DNA form the complex of 50～300 nanometers, and the cation high molecular material wherein and the mass ratio of DNA are 2 ~ 40:1.
6. by the described method of claim 5, it is characterized in that cation macromolecular material, hydrophilic polymer are connected with covalent manner with Angiopep-2 in the described macromolecule carrier.
7. by the described method of claim 5; It is characterized in that; Described macromolecule carrier prepares through following step: cation high molecular material and hydrophilic polymer are dissolved in pH value 7.8～8.2 phosphate buffers with the molecular proportion of 1:2 ~ 20, stirring at room reaction 1～3 hour, the group generation specific reaction that wherein contains; Generate cation high molecular material-hydrophilic polymer, ultrafiltration is removed unreacted hydrophilic polymer and is replaced by pH value 6.8～7.2 phosphate buffers; Simultaneously; Sulfydryl is introduced in Angiopep-2 and sulfhydrylization reagent reaction back; Generate cation high molecular material-hydrophilic polymer-lactoferrin after 12～36 hours with the molecular proportion of 1～10:1 and the special group stirring at room reaction on cation high molecular material-hydrophilic polymer, molecular exclusion chromatography is removed unreacted Angiopep-2.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110170055A (en) * | 2019-06-04 | 2019-08-27 | 哈尔滨理工大学 | A kind of preparation method of novel dual cerebral ischemia targeted nano carrier material |
| CN113117096A (en) * | 2019-12-30 | 2021-07-16 | 复旦大学 | NCAM mimic peptide modified pH sensitive gene bionic delivery system and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101160403A (en) * | 2005-02-18 | 2008-04-09 | 安吉奥开米公司 | Molecules for transporting a compound across the blood-brain barrier |
| CN101262890A (en) * | 2005-07-15 | 2008-09-10 | 安吉奥化学公司 | Use of aprotinin polypeptides as carriers in pharmaceutical conjugates |
-
2011
- 2011-02-01 CN CN2011100344941A patent/CN102614524A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101160403A (en) * | 2005-02-18 | 2008-04-09 | 安吉奥开米公司 | Molecules for transporting a compound across the blood-brain barrier |
| CN101262890A (en) * | 2005-07-15 | 2008-09-10 | 安吉奥化学公司 | Use of aprotinin polypeptides as carriers in pharmaceutical conjugates |
Non-Patent Citations (1)
| Title |
|---|
| WEILUN KE ET.AL: "Gene delivery targeted to the brain using an Angiopep-conjugated polyethyleneglycol-modified polyamidoamine dendrimer", 《BIOMATERIALS》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110170055A (en) * | 2019-06-04 | 2019-08-27 | 哈尔滨理工大学 | A kind of preparation method of novel dual cerebral ischemia targeted nano carrier material |
| CN113117096A (en) * | 2019-12-30 | 2021-07-16 | 复旦大学 | NCAM mimic peptide modified pH sensitive gene bionic delivery system and application thereof |
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Application publication date: 20120801 |