CN109384850A - Overall process target polypeptide and its application in building cancer target diagnosis and treatment delivery system - Google Patents
Overall process target polypeptide and its application in building cancer target diagnosis and treatment delivery system Download PDFInfo
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Abstract
The invention belongs to pharmaceutical fields, more particularly to a kind of overall process target polypeptide molecule with targeting brain capillary endothelial cell, endothelial cells in tumor neogenetic blood vessels, tumour mimicry blood vessel, tumour cell and tumor stem cell and its stablize optimization peptide molecule and its purposes of the compound, delivery system modified in tumor diagnosis and therapy.The present invention is prepared for the preparation of the drug and polymer carrier of overall process target polypeptide WVAP and WVAP modification, and its application in tumor imaging and targeted therapy are constructed with delivery system, as the result is shown: WVAP may span across blood brain barrier, target tumor new vessels and across bloodtumor barrier, target tumor mimicry blood vessel, tumour cell and tumor stem cell;Nanoscale medicine delivery system constructed by the polymer carrier of WVAP modification can be effectively by package-contained drug delivery to intracerebral, and target tumor tissue significantly improves antitumor drug effect.
Description
Technical field
The invention belongs to pharmaceutical fields, are related to overall process target polypeptide and application thereof, and in particular to have across blood brain barrier and
Across bloodtumor barrier, the overall process targeting of target tumor new vessels, tumour mimicry blood vessel, tumour cell and tumor stem cell
The nanoscale medicine delivery system of peptide molecule and its medicinal composition and modification.
Background technique
Prior art discloses tumor mortality rate, to be in all mortalities the first, be seriously threaten human life and
The disease of health.In clinical treatment, main means of traditional chemotherapy as tumor pharmacother exist and select tumor tissues
Property is poor, toxicity is big, therapeutic window is narrow, is also easy to produce the defects of multidrug resistance.It therefore, is the limitation for overcoming traditional treatment means, closely
Nian Lai, active targeting become the Critical policies for improving tumor tissues targeting efficiency.Active targeting strategy is mainly for tumor tissues
In highly expressed receptor or transporter, utilize the corresponding ligand that there is identification, binding ability with specific receptor or transporter, will
Drug or nanoscale medicine delivery system are delivered in tumor tissues or cell.Drug or nanometer after studies have shown that is ligand modified pass medicine
System can deliver the medicament to tumor group by the specific recognition of cell surface receptor or transporter and ligand, combination, internalization
It knits and into the cell, to realize the active targeting to tumour.According to data, current ligand is mostly just for a certain receptor
Or the targeting of a certain cell, however tumor group is woven with tumour cell, there are also tumor stem cells, tumour mimicry blood vessel, tumor neogenetic
Blood vessel and bloodtumor barrier (BTB) etc., there is also blood brain barrier (BBB) for brain tumor, make about 98% small molecule chemotherapeutic drug
Almost the macromolecular drugs such as 100% protide can not enter intracerebral through BBB, cause drug therapy nearly unavailable, therefore, open
It sends out and has important practical significance to the tumour ligand that especially brain tumor growth development overall process all has target function.
VAP(LVAP1 (L-type amino acid sequence is SNTRVAP) and its stable optimization polypeptideDVAP (D type amino acid sequenceDPDADVDRDTDNDS)、LVAP2 (L-type amino acid sequence is PAVRTNS) and its stable optimization polypeptideSVAP (D type amino acid sequenceDSDNDTDRDVDADP)) be the application verify research early period with glucose regulated protein GRP78 there is height to be combined active 7
Peptide, energy target tumor neovascular endothelium cell and leap BTB, targets neoplastic cells and tumor stem cell, have in vivo
Good homing capacity, but not across BBB.
WSW(L(L-type amino acid sequence is SYPGWSW's) to WSW and its stabilization optimizes polypeptideDWSW (D type amino acid sequenceDWDSDWDGDPDYDS)) and that verifies the application early period with quorum sensing receptor there is height to be combined active 7 peptide, can
It targets brain capillary endothelial cell and penetrates BBB, target tumor neovascular endothelium cell and penetrate BTB, and target tumor
Cell, but without tumor stem cell target function.
Based on state of the art and basis, present inventor is quasi- to be provided overall process target polypeptide and its swells in building
Tumor targets the application in diagnosis and treatment delivery system, makes more effectively to play to brain tumor or has brain metastes feature periphery tumour
Targeting diagnosis and treatment effect, and the delivery system drug combination that will contain tumor therapeutic agent plays synergistic antitumor work
With
Further existing target polypeptide molecule is transformed, using fusion protein principle, it is suitable that VAP and WSW is passed through
Bridge crosslinking structure (linker) be spliced into WVAP (LWVAP:PAVRTNS-linker-SYPGWSW (LWVAP1)、SNTRVAP-
linker-SYPGWSW(LWVAP2)、SYPGWSW-linker-PAVRTNS(L) and SYPGWSW-linker-SNTRVAP WVAP3
(LWVAP4);DWVAP:DSDNDTDRDVDADP-linker-DWDSDWDGDPDYDS(DWVAP1)、DPDADVDRDTDNDS-linker-DWDSDWDGDPDYDS(DWVAP2)、DWDSDWDGDPDYDS-linker-DSDNDTDRDVDADP(DWVAP3) andDWDSDWDGDPDYDS-
linker-DPDADVDRDTDNDS(DWVAP4);LDWVAP:PAVRTNS-linker-DWDSDWDGDPDYDS(LDWVAP1)、
SNTRVAP-linker-DWDSDWDGDPDYDS(LDWVAP2)、DWDSDWDGDPDYDS-linker-PAVRTNS(LDWVAP3)、DWDSDWDGDPDYDS-linker-SNTRVAP(LDWVAP4)、DSDNDTDRDVDADP-linker-SYPGWSW(LDWVAP5)、DPDADVDRDTDNDS-linker-SYPGWSW(LDWVAP6)、SYPGWSW-linker-DSDNDTDRDVDADP(LDWVAP7) and
SYPGWSW-linker-DPDADVDRDTDNDS(LDWVAP8)), possess it to brain tumor or have brain metastes feature periphery swollen
Tumor growth develops overall process target function.Meanwhile constructing the macromolecule of the diagnosing and treating medicinal composition of WVAP modification, modification
Carrier material and its constructed delivery system, to more effectively play to brain tumor or there is brain metastes feature periphery
The targeting diagnosis and treatment of tumour act on, and the WVAP delivery system for containing tumor therapeutic agent is carried out drug combination, play collaboration and increase
Imitate antineoplastic action.
Summary of the invention
The purpose of the present invention is being based on state of the art and basis, overall process target polypeptide is provided and its in building tumour
Target the application in diagnosis and treatment delivery system, and in particular to have across blood brain barrier and across bloodtumor barrier, target tumor is newborn
Blood vessel, tumour mimicry blood vessel, tumour cell and tumor stem cell overall process target polypeptide molecule and its medicinal composition and repair
The nanoscale medicine delivery system of decorations.
The present invention provides overall process target polypeptide WVAP and its height of the diagnosing and treating medicinal composition of modification, modification
The nanoscale medicine delivery systems such as molecular vehicle material and its constructed liposome, polymer micelle, polymer disc, nanoparticle, and
Its application in brain tumor or periphery diagnosing tumor and targeted therapy with brain metastes feature;And WVAP modification pass
Medicine system contains application of the tumor therapeutic agent in the synergy of drug combination is antitumor.
Specifically, blood brain barrier and bloodtumor barrier, and target tumor new vessels, tumour are crossed in of the invention having
The overall process target polypeptide molecule WVAP of mimicry blood vessel, tumour cell and tumor stem cell, will by bridge crosslinking structure (linker)
WSW and VAP be covalently attached and to be formed (LWVAP:PAVRTNS-linker-SYPGWSW (LWVAP1)、SNTRVAP-linker-
SYPGWSW(LWVAP2)、SYPGWSW-linker-PAVRTNS(L) and SYPGWSW-linker-SNTRVAP WVAP3
(LWVAP4);DWVAP:DSDNDTDRDVDADP-linker-DWDSDWDGDPDYDS(DWVAP1)、DPDADVDRDTDNDS-linker-DWDSDWDGDPDYDS(DWVAP2)、DWDSDWDGDPDYDS-linker-DSDNDTDRDVDADP(DWVAP3) andDWDSDWDGDPDYDS-
linker-DPDADVDRDTDNDS(DWVAP4);LDWVAP:PAVRTNS-linker-DWDSDWDGDPDYDS(LDWVAP1)、
SNTRVAP-linker-DWDSDWDGDPDYDS(LDWVAP2)、DWDSDWDGDPDYDS-linker-PAVRTNS(LDWVAP3)、DWDSDWDGDPDYDS-linker-SNTRVAP(LDWVAP4)、DSDNDTDRDVDADP-linker-SYPGWSW(LDWVAP5)、DPDADVDRDTDNDS-linker-SYPGWSW(LDWVAP6)、SYPGWSW-linker-DSDNDTDRDVDADP(LDWVAP7) and
SYPGWSW-linker-DPDADVDRDTDNDS(LDWVAP8));
In the present invention, with WVAP modified medicaments molecule and polymer carrier, WVAP- medicinal composition, WVAP are constructed
The nanoscale medicine delivery system of modification can improve drug to brain, brain tumor or targeting diagnosis and treatment with brain metastes feature periphery tumour
Effect.The WVAP delivery system for containing tumor therapeutic agent is further subjected to drug combination, is realized synergistic antitumor
Effect.
More specifically, the present invention utilizes fusion protein principle, designs and is prepared for peptide molecule WVAP, make it while having
The targeting ability of WSW and VAP can be played across blood brain barrier and bloodtumor barrier, and target tumor new vessels, tumour mimicry
The especially brain tumor growth of the tumour of blood vessel, tumour cell and tumor stem cell develops overall process targeting.
In the present invention, the splicing of WSW and VAP are covalently attached by suitable bridge crosslinking structure, and the bridge crosslinking structure used is ammonia
Base fatty acid and its derivative, such as cysteine, aminocaproic acid;Amino aromatic acid and its derivative, such as p-aminobenzoic acid
Deng;Or oligopeptides, such as GG, GSSG.
WVAP designed by the present invention can further introduce active function groups in the molecule, and the drug for constructing its modification is compound
Object, polymer carrier, the site for introducing functional group is nitrogen end, carbon teminal or the bridge crosslinking structure of WVAP, preferably bridge crosslinking structure, is made
The molecule forms Y-shaped structure, is conducive to the performance of target function.
After WVAP designed by the present invention introduces cysteine, sulfydryl in its molecule and maleimide amino-functionalization can be utilized
Image substance (such as Fluorescein, nir dye Cy5.5, IR820, DiR, nuclear magnetic resonance image agent Gd-DTPA, irradiation image
Agent99mTc-DTPA etc.) it reacts and forms compound.
WVAP modified medicaments designed by the present invention, including react to form pH sensitivity by the own hydrazine derivate of maleimide
Hydrazone bond (being related to the drug containing ketone or aldehyde radical such as adriamycin, Epi-ADM) passes through 3- (2- pyridine dimercapto) propanoic derivatives
Reaction formed disulfide bond (be related to taxol, Docetaxel, Cabazitaxel, camptothecine, hydroxycamptothecin, 9-nitrocamptothecin,
The drug of the hydroxyls such as Irinotecan, vincristine and vinorelbine or amino) or pass through boric acid base group in dopamine and drug
Reaction forms pH sensitivity boric acid rouge (being related to the drug of the boracics acid groups such as bortezomib) or directly forms acyl by synthesis in solid state
The WVAP- medicinal composition of amine key (being related to the polypeptide drugs such as p53 activating peptide).
After WVAP designed by the present invention introduces cysteine, the poly- second two in the base of amine functions containing maleimide can be modified
Alcohol-distearoylphosphatidylethanolamine (PEG-DSPE), polyethylene glycol-polylactic acid (PEG-PLA), polyethylene glycol-lactic acid hydroxyl
On the polymer carriers such as acetic acid copolymer (PEG-PLGA), polyethylene glycol-polycaprolactone (PEG-PCL), it can be used for
The building of the nanoscale medicine delivery systems such as liposome, polymer micelle, polymer disc, the nanoparticle of WVAP modification.
The nanoscale medicine delivery system of the modification of WVAP designed by the present invention can contain the anthracycline drugs such as adriamycin and Epi-ADM
The taxones such as object, taxol and Docetaxel and Cabazitaxel, camptothecine and hydroxycamptothecin and 9- nitro camplotheca acuminata
Vincas drug, bortezomib and the Ka Feizuo such as the camptothecines such as alkali and Irinotecan, vincristine and vinorelbine
The polypeptide toxins such as lactones drug, p53 activating peptide, melittin and the scorpion venom peptides such as proteasome inhibitors, the parithenolides such as rice,
The anti-tumor drugs such as antibacterial peptide;Image substance can also be contained, such as coumarin 6, FAM, nir dye Cy5.5, IR820, DiR,
DiD, nuclear magnetic resonance image agent Gd-DTPA etc..
WVAP designed by the present invention can mediate drug or nanoscale medicine delivery system cross over blood brain barrier and bloodtumor barrier,
Target tumor new vessels, tumour mimicry blood vessel, tumour cell and tumor stem cell turn for brain, brain tumor or with brain
Move targeting diagnosis and the treatment of feature periphery tumour.
The delivery system of the modification of WVAP designed by the present invention contains tumor therapeutic agent (such as adriamycin, Epi-ADM, purple
China fir alcohol, Docetaxel, Cabazitaxel, camptothecine, hydroxycamptothecin, 9-nitrocamptothecin, Irinotecan, vincristine, length
The polypeptide toxins, antibacterial peptide such as spring Rui Bin, bortezomib, Carfilzomib, parithenolide, p53 activating peptide, melittin and scorpion venom peptide
Deng), with other tumor therapeutic agents (such as Temozolomide, cyclophosphamide, Etoposide, mercaptopurine, gemcitabine, cytarabine,
5 FU 5 fluorouracil, Teniposide, Epothilones, actinomycin D, mitoxantrone, mitomycin, bleomycin, for Buddhist nun's class drug,
Platinum medicine, bevacizumab, trastuzumab etc.) drug combination, to realize synergistic antitumor effect.
The delivery system of the modification of WVAP designed by the present invention contains different tumor therapeutic agent drug combinations, such as contains
Inhibit adriamycin, Epi-ADM, taxol, Docetaxel, Cabazitaxel, camptothecine, the hydroxy-camptothecin of growth of tumour cell
The delivery system of the tumor therapeutic agents such as alkali, 9-nitrocamptothecin, Irinotecan, vincristine, vinorelbine, has with containing
The delivery system of the tumor therapeutic agents such as bortezomib, Carfilzomib, the parithenolide of resisting tumour stem cells effect, which is combined, to be used
Medicine, or it is swollen with polypeptide toxins, the antibacterial peptides such as polypeptide p53 activating peptide, melittin and scorpion venom peptide with antitumor action etc. is contained
The delivery system drug combination of tumor therapeutic agent, to realize synergistic antitumor effect.
In the present invention, following experiment has been carried out:
1) synthesis of WVAP, WVAP-Cys and its fluorescent marker (WVAP-Fluorescein, WVAP-Cy7),
WVAP and WVAP-Cys is prepared using solid phase synthesis process;Pass through the Michael of maleimide base group and sulfydryl
Addition reaction has synthesized WVAP-Fluorescein or WVAP-Cy7;MS characterizes structure.
2) WVAP-DTPA-Gd and WVAP-DTPA-99mThe synthesis of Tc
Synthesized WVAP-DTPA with the Michael addition reaction of sulfydryl by maleimide base group, chelate Gd or99mTc
Obtain WVAP-DTPA-Gd or WVAP-DTPA-99mTc。
3) the compatibility evaluation of WVAP and GRP78
Using the binding constant K of surface plasma resonance method (SPR) measurement WVAP peptide and GRP78 receptor proteinDValue, is commented
Its compatibility of valence.
4) WVAP estimation of stability
Investigate stability of the WVAP in serum;WVAP and mice serum are incubated at 37 DEG C, in different time points
The concentration of detection polypeptide evaluates its stability.
5) WVAP targeting merit rating
WVAP-Fluorescein is investigated to brain capillary endothelial cell (BCEC), huve cell (HUVEC)
With the targeting of Model Tumor Cells (such as: brain glioblastoma cell U87);External 3D tumour spherical model is investigated to WVAP-
The intake ability of Fluorescein.
6) WVAP- medicinal composition is prepared
WVAP after introducing cysteine is reacted with the own hydrazine derivate of the maleimide of drug, forms the hydrazone bond of sensitivity containing pH
Polypeptide-medicinal composition, wherein involved drug includes the drug containing ketone or aldehyde radical such as adriamycin, Epi-ADM;
WVAP after introducing cysteine is reacted with 3- (2- pyridine dimercapto) propanoic derivatives of drug, is formed and is contained two sulphur
Polypeptide-medicinal composition of key, wherein involved drug includes taxol, Docetaxel, Cabazitaxel, camptothecine, hydroxyl
The drug of the hydroxyls such as camptothecine, 9-nitrocamptothecin, Irinotecan, vincristine, vinorelbine or amino;
WVAP is reacted by modifying upper dopamine with the boric acid base group of drug, forms the more of the boric acid of sensitivity containing pH rouge
Peptide-drug compound, wherein involved drug includes the drug of the boracics acid groups such as bortezomib;
WVAP is directly condensed with polypeptide drugs by synthesis in solid state, wherein involved drug includes the polypeptides such as p53 activating peptide
Drug.
7) building and characterization of the nanoscale medicine delivery system of WVAP modification
First synthesis WVAP modification high molecular material WVAP-PEG-DSPE, WVAP-PEG-PLA, WVAP-PEG-PLGA,
WVAP-PEG-PCL etc..Upper free sulfhydryl groups of WVAP by introducing cysteine and Mal-PEG-DSPE, Mal-PEG-PLA,
The synthesis of material is realized in the reaction of maleimide contained by Mal-PEG-PLGA, Mal-PEG-PCL etc., it may be assumed that by Mal-PEG-
DSPE, Mal-PEG-PLA, Mal-PEG-PLGA, Mal-PEG-PCL etc. are dissolved in acetonitrile respectively, rotary evaporation, and film forming adds
PBS (pH8.0) reaction for entering the WVAP containing sulfydryl is preparedDThe high molecular material of WVAP modification;
Then the nanoscale medicine delivery system of WVAP modification is prepared.A certain amount of WVAP-PEG-DSPE and mPEG-DSPE and phosphatide
With cholesterol or WVAP-PEG-DSPE and mPEG-DSPE or WVAP-PEG-PLA and mPEG-PLA or WVAP-PEG-PLGA
With mPEG-PLGA or WVAP-PEG-PCL and mPEG-PCL and a certain amount of said medicine, divided using the methods of film forming aquation
It does not prepare correspondingDThe nanometers such as WVAP modified liposome, polymer micelle, polymer disc, polymer nanoparticle, which are passed, wants system;
Laser diffraction particle size instrument levies nanoscale medicine delivery system partial size and particle diameter distribution.
8) the inside and outside tumor-targeting evaluation of the nanoscale medicine delivery system of WVAP modification
It investigates U87 cell, HUVEC cell and U87 tumour sphere external model and nanometer is modified to the WVAP for containing fluorescent material
The intake situation of delivery system;
Nanometer, which is modified, by the WVAP that lotus U87 subcutaneous transplantation knurl model nude mice tail vein injection contains fluorescent material passs medicine system
System, investigates it and is distributed in the tumour at each time point.
9) the inside and outside antitumous effect evaluation of the nanoscale medicine delivery system of WVAP modification
The WVAP modification nanoscale medicine delivery system for containing tumor therapeutic agent is investigated with mtt assay to U87 cell and HUVEC cell
Growth in vitro inhibitory effect;It is repaired by the WVAP that lotus U87 primary tumor model nude mice tail vein injection contains tumor therapeutic agent
Nanoscale medicine delivery system is adornd, is to refer to life span, tumor tissue cell's apoptosis, new vessels, mimicry blood vessel and stem cell population
Mark evaluates internal antitumous effect.
10) drug combination is tested
Nanometer is modified by the WVAP that lotus U87 Orthotopic Transplantation Model nude mice tail vein injection contains tumor therapeutic agent to pass
The above-mentioned clinical common tumor therapeutic agent drug combination such as medicine system and Temozolomide;Contain the WVAP modification of tumor therapeutic agent
Nanoscale medicine delivery system with contain passing for the tumor therapeutic agents such as bortezomib, parithenolide with resisting tumour stem cells effect
The system combined medication of medicine;It contains the WVAP modification nanoscale medicine delivery system of tumor therapeutic agent and contains with antitumor action
The polypeptide drugs drug combinations such as polypeptide toxins, the antibacterial peptides such as p53 activating peptide, melittin and scorpion venom peptide are to refer to median survival interval
Mark evaluation antitumous effect.
It is investigated the present invention provides WVAP preparation and property and above-mentioned modified medicinal composition and nanometer passs medicine system
System is used for the material base of tumour diagnosis and treatment, through testing, the results showed that WVAP is provided simultaneously with the targeting ability of WSW and VAP, in mould
There is good brain and tumor tissues targeting ability and image effect in type animal body, and show more excellent brain tumor targeting energy
Power;The nanoscale medicine delivery system of WVAP modification shows good cancer target performance and stronger anti-brain tumor effect;Packet
The delivery system for carrying the WVAP modification of tumor therapeutic agent contains not with other tumor therapeutic agent drug combinations or delivery system
Same tumor therapeutic agent drug combination has apparent synergistic antitumous effect.
Detailed description of the invention
Fig. 1,DThe ESI-MS map of WVAP1-Cys,
It wherein shows, ESI-MS:1822.4 is consistent with theoretical molecular weight.
Fig. 2,DThe ESI-MS map of WVAP1-Fluorescein,
It wherein shows, ESI-MS:2250.4 is consistent with theoretical molecular weight.
Fig. 3,DThe ESI-MS map of WVAP1-Cy7,
It wherein shows, ESI-MS:2517.8 is consistent with theoretical molecular weight.
Attached drawing 4,DWVAP1-PEG-PLA's1H-NMR map,
Wherein, the nuclear magnetic spectrum 7.0ppm of Mal-PEG-PLA shows maleimide peak, andDWVAP1-PEG-PLA's
The peak disappears in nuclear magnetic spectrum, shows the maleimide base group fully reacting in Mal-PEG-PLA.
Fig. 5,DThe combination activity of WVAP1 and GRP78 albumen,
Which showDThe combination activity of WVAP1 and GRP78 is high, withDVAP is similar, andDWSW and GRP78 albumen is not tied
It closes,DThe K of WVAP1DValue is 0.686 μM, withDVAP is quite (0.102 μM).
Fig. 6,DThe serum stability of WVAP1,
Wherein, figure ordinate is the residual percentage of complete polypeptide, it is seen that polypeptide has very high in 50% mice serum
Stability, be incubated for for 24 hours after it is almost non-degradable.
Fig. 7, rat primary brain capillary endothelial cell BCEC, human glioma cell U87 and human umblilical vein endothelial are thin
Intake of the born of the same parents HUVEC to Fluorescein labeling polypeptide,
Wherein, figure A is Fluorescein labelDWVAP1、DWSW、DVAP and free fluorescein and BCEC cytosis
12h, respectively with the laser co-focusing photo after U87 cell and HUVEC cytosis 4h;Scheming B, figure C and figure D is respectively with loading
The cellular uptake result that flow cytometer measures after product and BCEC cell, U87 cell and HUVEC cytosis, it is seen that BCEC is thin
Born of the same parents coupleDWVAP1 andDThe intake of WSW be apparently higher than free fluorescein andDVAP is rightDWVAP1 andDThe intake of WSW is without obvious poor
It is different;U87 cell and HUVEC cell have preferable intake to three polypeptides.
Fig. 8,DWVAP1-Cy7's and load DiRDThe polymer micelle of WVAP modification distribution and normal mouse brain in tumor in situ
Interior distribution,
Wherein, figure A is the fluorescence distribution image after lotus U87 primary tumor nude mice tail vein injection Cy7 labeling polypeptide 1h, schemes B
For various time points brain fluorescence semi-quantitative results after administration, show: Cy7 labelDWVAP1、DWSW andDVAP is in tumour
Accumulation is all remarkably higher than free fluorescein Cy7 (p < 0.001 * * *), and cancer target effectDWVAP1-Cy7>DWSW-Cy7>DVAP-Cy7;Scheming C is the fluorescence distribution image after lotus U87 primary tumor nude mice tail vein injection carries DiR polymer micelle for 24 hours, schemes D
For the fluorescence semi-quantitative results that in vitro brain tumor rear for 24 hours is administered, figure E is four kinds of different polymer micelles in normal mouse brain
The distribution situation in portion, it is shown thatDWVAP1 modification micella, which has, not to be weaker thanDThe polymer micelle of WSW modification enters brain effect;Scheme F
For administration 2h and for 24 hours the fluorescent quantitation of back brain tumour as a result, showingDThe polymer micelle of WVAP1 modification can be targeted preferably
To tumor locus.
Fig. 9,DExternal blood brain barrier, bloodtumor barrier transhipment and the tumour ball of the polymer micelle of WVAP1 modification are taken the photograph
It takes, wherein
Scheme A and scheme B to be respectively to carry coumarin 6DWVAP1 modification polymer micelle (DWVAP1-Micelle/C6) with it is right
Product in the same old wayDWSW modification polymer micelle (DWSW-Micelle/C6)、DVAP modification polymer micelle (DVAP-Micelle/
C6 it) is transported in different time points with polymer micelle (Micelle/C6) and crosses over external blood brain barrier (BBB) and blood-tumour screen
Hinder the percentage of (BTB) model, it is shown that: in 30min, 1,2 and 4h,DWVAP1-Micelle/C6 andDWSW-Micelle/C6
Be transported through external BBB model amount be apparently higher than no target Micelle/C6 andDVAP-Micelle/C6, and the transhipment hundred of the two
Divide rate without significant difference;In 30min, 1,2 and 4h,DWVAP1-Micelle/C6、DWSW-Micelle/C6 andDVAP-
The amount that Micelle/C6 is transported through external BTB model is apparently higher than no target Micelle/C6, and the transhipment percentage of three without
Significant difference;Scheming C is to absorb situation across three-dimensional nodule ball after BBB or BTB, it is shown that:DWVAP1-Micelle/C6 andDWSW-
Micelle/C6 can be absorbed effectively across BBB or BTB and by lower room U87 three-dimensional nodule ball,DVAP-Micelle/C6 may span across
BTB is simultaneously absorbed by lower room U87 three-dimensional nodule ball, but lacks the ability for crossing over BBB.
Figure 10, the partial size for carrying taxol and parithenolide polymer micelle,
Wherein, taxol is respectively carriedDWVAP modification polymer micelle (DWVAP1-Micelle/PTX) and carry little Bai
Chrysanthemum lactonesDWVAP1 modification polymer micelle (DWVAP1-Micelle/PTL partial size picture), it is shown that,DWVAP1-
Micelle/PTL andDThe partial size of WVAP1-Micelle/PTX is respectively 45nm and 30nm, and particle diameter distribution is narrow.
It is bent that Figure 11, load taxol and parithenolide polymer micelle inhibit U87 cell and HUVEC cell to grow in vitro
Line, wherein
Figure A and figure B be respectively Taxol (taxol), Micelle/PTX,DWVAP1-Micelle/PTX inhibit U87 cell and
HUVEC cell growth curve, after figure A shows U87 cell administration 4h, culture 72h, IC50Respectively 2.87,1.09 and
0.342 μM,DThe external activity of WVAP1-Micelle/PTX is 3.18 times of Micelle/PTX;Figure B show HUVEC cell to
After medicine 4h, culture 72h, IC50Respectively 1.173,0.749 and 0.267 μM,DThe external activity of WVAP1-Micelle/PTX is
2.81 times of Micelle/PTX;Figure C and figure D be respectively PTL (free parithenolide), Micelle/PTL,DWVAP1-
After Micelle/PTL inhibits U87 cell and HUVEC cell growth curve, figure C to show U87 cell administration 4h, culture 72h,
IC50Respectively 19.13,15.08 and 7.59 μM,DThe external activity of WVAP1-Micelle/PTL is the 1.99 of Micelle/PTL
Times;After figure D shows HUVEC cell administration 4h, culture 72h, IC50Respectively 5.670,3.828 and 2.766 μM,DWVAP1-
The external activity of Micelle/PTL is 1.38 times of Micelle/PTL.
Figure 12, load taxol and parithenolide polymer micelle inhibit new vessels and mimicry vascularization in vitro,
Wherein, figure PTL, Taxol,DWVAP1-Micelle/PTX andDWVAP1-Micelle/PTL is to new vessels (A)
With the inhibition photo of mimicry blood vessel (B) external model, compared to free PTL and Taxol,DWVAP1-Micelle/PTX、DWVAP1-Micelle/PTL inhibits the formation of new vessels and mimicry blood vessel more significant.Figure 13, taxol polymer micelle is carried
The model nude mice survivorship curve of anti-glioma,
Wherein show, physiological saline group, Taxol group, Micelle/PTX group,DVAP-Micelle/PTX group,DWSW-
Micelle/PTX group,DThe WVAP1-Micelle/PTX group mean survival time is respectively 19,20,21,23.5,27.5 and 29.5
It, the results showed that, compared with remaining each group,DWVAP1-Micelle/PTX can significantly extend the existence of Brain Glioma Model nude mice
Time (n=8).
Figure 14, the model nude mice survivorship curve for carrying the anti-glioma of parithenolide polymer micelle,
Wherein show, physiological saline group, PTL group, Micelle/PTL group,DWVAP1-Micelle/PTL group the average survival time
Time is respectively 29.5,32,32 and 37 days, the results showed that, compared with remaining each group,DWVAP1-Micelle/PTL can significantly prolong
The life span (n=8) of long Brain Glioma Model nude mice.
The model nude mice existence that Figure 15, load parithenolide and taxol polymer micelle are administered in combination anti-glioma is bent
Line,
Wherein show, physiological saline group,DWVAP1-Micelle/PTL group,DWVAP1-Micelle/PTX group,DWVAP1-
Micelle/PTL withDThe mean survival time of WVAP1-Micelle/PTX administering drug combinations group is respectively 33.5,47.5,51.5 and
74 days, the results showed that, compared with remaining each group,DWVAP1-Micelle/PTL withDWVAP1-Micelle/PTX administering drug combinations can
The significant life span (n=8) for extending Brain Glioma Model nude mice.
Figure 16, the model nude mice existence that parithenolide polymer micelle is administered in combination anti-glioma with Temozolomide is carried
Curve,
Wherein show, physiological saline group,DWVAP1-Micelle/PTL group, TMZ (Temozolomide) group,DWVAP1-
The mean survival time of Micelle/PTL and TMZ administering drug combinations group is respectively 29.5,37,39 and 59 days, the results showed that, with it
Remaining each group is compared, and the life span (n=8) that can significantly extend Brain Glioma Model nude mice is administered in combination.
Figure 17, TUNEL coloration result
Wherein, figure A be physiological saline group, TMZ group,DWVAP1-Micelle/PTX group,DWVAP1-Micelle/PTL group,DWVAP1-Micelle/PTL and TMZ administering drug combinations group,DWVAP1-Micelle/PTL withDWVAP1-Micelle/PTX combine to
Medicine group leads to the TUNEL stained photographs (bar=50 μm) of glioma apoptosis, wherein the positive nucleus of apoptosis in brown color or
Sepia, figure B are quantitative result.
The bis- coloration results of Figure 18, CD31/PAS,
Wherein, figure A be physiological saline group, TMZ group,DWVAP1-Micelle/PTX group,DWVAP1-Micelle/PTL group,DWVAP1-Micelle/PTL and TMZ administering drug combinations group,DWVAP1-Micelle/PTL withDWVAP1-Micelle/PTX combine to
The bis- stained photographs of CD31/PAS (bar=100 μm) that medicine group inhibits new vessels to be formed, wherein new vessels nucleus is in pale brown
Color or sepia, figure B are quantitative result.
Figure 19, CD133 coloration result,
Wherein, figure A be physiological saline group, TMZ group,DWVAP1-Micelle/PTX group,DWVAP1-Micelle/PTL group,DWVAP1-Micelle/PTL and TMZ administering drug combinations group,DWVAP1-Micelle/PTL withDWVAP1-Micelle/PTX combine to
Medicine group kill tumor stem cell CD133 stained photographs (bar=100 μm), wherein tumor stem cell be in brown color or sepia,
Figure B is quantitative result.
Specific embodiment
It will be helpful to further understand the present invention by following embodiments, but the present invention is not limited to range described below.
Embodiment 1
DWVAP1-Cys、DWVAP1-Fluorescein、DWVAP1-Cy7、DWVAP1- drug,DThe conjunction of WVAP1-PEG-PLA
At with characterization
1)DThe synthesis and characterization of WVAP1-Cys
Using solid phase polypeptide synthesis, synthesisD(sequence is WVAP1-CysDSDNDTDRDVDADPDC- aminocaproic acid-DWDSDWDGDPDYDS), specific method: sequentially column are sequentially ingressed into amino acid on Boc-Cys PAM resin, are with HBTU/DIEA
Condensing agent, TFA are that deprotection agent is reacted.After the reaction was completed, resin is cut with the hydrogen fluoride containing P-cresol, ice
Bath is stirred to react 1h.Decompression pumps hydrogen fluoride in pipe after reaction, and ice ether precipitates and washs precipitating 3 times, precipitates with 20%
Acetonitrile re-dissolves, and rotates after collecting filtrate, obtains polypeptide crude product solution, polypeptide crude product acetonitrile/water (containing 0.1%TFA) body
System isolates and purifies.ESI-MS characterizes molecular weight (Mw), and mass spectrogram is as shown in Figure 1;
2)DWVAP1-Fluorescein withDThe synthesis and characterization of WVAP1-Cy7
Above-mentioned steps are obtainedDWVAP1-Cys is dissolved in the PBS solution of 0.1M (pH7.2), takes Fluorescein-5-
Maleimide or Cy7-maleimide is dissolved in DMF, and magnetic agitation is reacted after the two mixing, HPLC monitoring, toDWVAP-Cys is anti-
It should stop reaction afterwards completely, preparation liquid phase purifying is isolated and purified, is freeze-dried with acetonitrile/water (containing 0.1%TFA) systemDWVAP-Fluorescein orDWVAP1-Cy7 sterling, mass spectrogram are as shown in Figure 2,3;
3) it preparesDWVAP1-DTPA-Gd withDWVAP1-DTPA-99mTc
It willDWVAP1-Cys is dissolved in the PBS solution of 0.1M (pH7.2), and maleimide-DTPA is dissolved in DMF, the two mixing
Magnetic agitation is reacted afterwards, HPLC monitoring, after complete reaction through preparing liquid phase, is separated with acetonitrile/water (containing 0.1%TFA) system
Purifying, is freeze-driedDWVAP1-DTPA sterling, chelate Gd or99mTc to obtain the final productDWVAP1-DTPA-Gd orDWVAP1-DTPA-99mTc;
4, it preparesDWVAP1- medicinal composition
WithDWVAP1- adriamycin composite prepares conductDEmbodiment of the WVAP1 connection containing ketone or aldehyde radical drug, 9.4mgDWVAP1-Cys polypeptide is dissolved in phosphate 3mL buffer (0.1mM, pH 7.0), and three (2- carboxyethyls) of 10 times of moles are added
Then in 4 DEG C of stirring 20min the own hydrazine derivate of adriamycin -6- maleimide of 4 times of moles is added, in room in phosphine (TCEP)
Temperature is protected from light 1h, and reaction solution preparation liquid phase is purified, is freeze-driedDWVAP1- adriamycin composite;
WithDWVAP1- taxol compound conductDWVAP1 connects the embodiment of hydroxyl or amino drug by disulfide bond.
200mg taxol is dissolved in 10mL chloroform, is cooled to 0-5 DEG C, and 40mg DCC and 60.4mg 3- (two mercapto of 2- pyridine is successively added
Base) propionic acid, after charging, it is warmed to room temperature reaction overnight.Reaction solution filtering, through column chromatographic purifying (CHCl3/MeOH=50:1-
15:1, V/V are eluted) obtain taxol -3- (2- pyridine dimercapto) propanoic derivatives, taxol -3- (2- pyridine dimercapto) propionic acid
Derivative is dissolved in 5mL DMF, 1.5 times of molesDWVAP1-Cys is dissolved in PBS/DMF, and solution ph holding 4~
5, taxol -3- (2- pyridine dimercapto) propanoic derivatives are added dropwise in polypeptide solution, in room temperature reaction 6h, through preparing liquid phase
Purifying is lyophilizedDWVAP1- taxol compound;
WithDWVAP1- boron is for assistant miaow compound conductDWVAP1 polypeptide connects the embodiment of boracic acid groups drug, according toDThe synthesis of WVAP1 is sequentially ingressed into amino acid on resin, and all amino acid residues access to polypeptide finishes, and trifluoroacetic acid is de-
The Boc of nitrogen end is gone to protect.The DMF solution of the succinic anhydride and DIEA that contain 3 times of moles is added, in room temperature reaction 30min.Washing
After resin, the trim,ethylchlorosilane protection dopamine of 5 times of moles is added, and using HBTU/DIEA as condensing agent, in room temperature reaction
1h, resin are cut with HF, and are purified through preparative HPLCDWVAP1- DOPA amine derivative, in the buffer of pH7.4,DWVAP1- DOPA amine derivative is mixed for assistant miaow with molar ratio 1:1 with boron to obtain the final productDWVAP1- boron is for assistant miaow compound;
WithDA kind of WVAP1-PMI (p53 activating peptide) conductDThe embodiment of WVAP1 connecting peptides drug, directly passes through solid phase
Polypeptide synthesis is made, comprising: determinesDAfter WVAP1-PMI polypeptide sequence, press and preparationDThe identical method of WVAP1 is sequentially ingressed into
Amino acid cuts through HF and obtains after purificationDWVAP1-PMI compound;
5)DThe synthesis and characterization of WVAP1- high molecular material compound
Pass throughDThe upper free sulfhydryl groups of WVAP1 are reacted with maleimide contained by Mal-PEG-PLA as preparationDWVAP1- high score
40mg Mal-PEG-PLA is dissolved in 5mL acetonitrile, rotary evaporation by the embodiment of sub- material composite, and 3mL is added in film forming
PBS (pH 8.0,0.2M) forms micella in 37 DEG C of aquations, and 9.6mg is added in 8hDWVAP1-Cys, overnight, HPLC is detected for reaction
Reaction, it is excessiveDWVAP1-Cys is removed by dialysis, freeze-drying;1H-NMR characterizes (as shown in Figure 4).
Embodiment 2DThe combination activity experiment of WVAP1 and glucose regulated protein GRP78
Pre- binding analysis is carried out by biacore system, selection pH5.0 is best GRP78 pH in conjunction with CM5 chip, will
Recombined human GRP78 is coupled on CM5 chip, and RU value reaches target value, willDWVAP1 is configured to the sample solution of various concentration, from
Low to high successively sample introduction is analyzed with Biacore T200Evaluation software softwareDThe combination of WVAP1 and albumen is living
Property, calculate separately its KDValue, and withDVAP andDWSW compares (as shown in Figure 5).
Embodiment 3DThe serum stability of WVAP1 investigates experiment
It willDWVAP1 is made into 1mg/mL aqueous solution, and 0.1mL is taken to be added in 25% mice serum of 0.9mL, 37 DEG C of incubations, point
Not in 0 and 15min, 0.5,1,2 and 4h takes out 100 μ L reaction solutions, is added albumen in 20 μ L trichloroacetic acids (TCA) precipitating serum, and 4
DEG C stand 20min, 12000rpm be centrifuged 10min, take 20 μ L of supernatant carry out HPLC analysis, serum stability result is (such as Fig. 6 institute
Show) showDWVAP1 has good serum stability.
Embodiment 4DThe cell in vitro targeting confirmatory experiment of WVAP1
1)DTargeting of the WVAP1 to brain capillary endothelial cell
Brain is taken after 4 week old SD rats broken end, is rapidly separated to obtain cerebral cortex in the D-Hanks solution of Yu Bingleng, is removed
It is shredded after meninx and the big blood vessel of brain, after 37 DEG C of digestion 90min of clostridiopetidase A and DNA enzymatic are added, 1000 revs/min of centrifugation 8min,
It discards supernatant, is transferred in 20% BSA, 1000g/min, 4 DEG C of centrifugation 20min discard liquid at the middle and upper levels, by bottom capilary
It is transferred in training liquid, 1000 revs/min of centrifugation 5min, it is outstanding to be made into capilary section with the DMEM culture solution containing 20% fetal calf serum
Liquid is inoculated in the burnt ware of copolymerization or 12 orifice plates, 37 DEG C, 5%CO2And cultivated under the conditions of saturated humidity for 24 hours, it changes into mould containing purine
The endothelium special culture solution of element continues after cultivating 72h, then changes the endothelium special culture solution culture containing Porcine HGF into
72h obtains primary brain capillary endothelial cell;With containing 10% fetal calf serum DMEM culture solution compound concentration be 5 μM FAM,DWVAP1-Fluorescein、DVAP-Fluorescein andDWSW-Fluorescein solution.Culture solution in culture plate is inhaled
Out, it is separately added into above-mentioned solution, 37 DEG C of incubation 12h inhale and abandon supernatant;It is washed three times with PBS solution, formaldehyde fixer is fixed thin
Born of the same parents, after DAPI carries out nuclear targeting, laser co-focusing observation, cell internalizing photo is as shown in Figure 7 A, is separately washed three times with PBS
Afterwards, flow cytometry analysis is carried out, as a result as shown in Figure 7 B;
2)DWVAP1 tests the targeting of human glioma cell U87
The brain glioblastoma cell (U87 cell) of the monolayer cultivation of logarithmic growth phase, with 0.25% trypsin digestion list
Layer culture cell, is made into single cell suspension with the DMEM culture solution containing 10% fetal calf serum, with every hole 1 × 105A cell inoculation
In 12 well culture plates, every pore volume 1mL is moved into culture plate in carbon dioxide incubator, 37 DEG C, 5%CO2And saturated humidity
Under the conditions of cultivate for 24 hours after, with containing 10% fetal calf serum DMEM culture solution compound concentration be 5 μM FAM,DWVAP1-
Fluorescein、DVAP-Fluorescein andDWSW-Fluorescein solution;Culture solution in culture plate is sucked out, respectively
Above-mentioned solution is added, 37 DEG C of incubation 4h inhale and abandon supernatant.Washed three times with PBS solution, the fixed cell of formaldehyde fixer, DAPI into
After row nuclear targeting, laser co-focusing is observed, and cell internalizing photo is as shown in Figure 7 A;After separately being washed three times with PBS, streaming is carried out
Cytometric Analysis, as a result as seen in figure 7 c;
3)DWVAP1 tests the targeting of Human umbilical vein endothelial cells HUVEC
The Human umbilical vein endothelial cells (HUVEC cell) of the monolayer cultivation of logarithmic growth phase, are ibid tested, cell internalizing
Photo is as shown in Figure 7 A, and flow cytometry analysis result is as illustrated in fig. 7d.
Embodiment 5DThe verifying of WVAP1 polypeptide in-vivo tumour targeting
Establish Brain Glioma Model mouse in situ: the U87 cell of logarithmic growth phase, every nude mouse inoculation 5 × 105It is a thin
Born of the same parents (are scattered in 5 μ L PBS buffer solution), after nude mouse anesthesia, are fixed with stereotaxic apparatus, and cell inoculation is in the corpus straitum right side
Portion (0.6mm, side 1.8mm, deep 3mm before bregma), routine observation nude mouse state carry out for the 5th day after nude inoculation tumour cell
Test, with 0.15 μm of oL/ only dosage by Cy7,DWVAP1-Cy7、DWSW-Cy7 andDVAP-Cy7 solution is injected by tail vein
In tumor bearing nude mice animal model body, 15,30,45 and 120min is respectively in living imaging instrument record fluorophor points after injection
Cloth, conventional treatment nude mice after 2h, takes brain tumor, with the fluorescence distribution (as shown in Figure 8 A) of living imaging instrument detection tumour, and
It carries out fluorescence sxemiquantitative calculating (as shown in Figure 8 B).
Embodiment 6DThe polymer micelle targeting verifying of WVAP1 modification
1) preparation carries Coumarin-6 polymer micelle
Weigh 1mgDWVAP1-PEG-PLA, 9mg mPEG-PLA and 5 μ g C6, are dissolved in 2mL acetonitrile, in 37 DEG C of water
Bath decompression (~0.085MPa) revolving film forming, room temperature in vacuo are dried overnight, and 2mL physiological saline aquation is added, and CL-4B column chromatography removes
Free C6 is removed, is madeDWVAP1-Micelle/C6,4 DEG C are kept in dark place, spare;
2)DWVAP1-Micelle is investigated across external blood brain barrier ability
Brain is taken after 4 week old SD rats broken end, is rapidly separated to obtain cerebral cortex in the D-Hanks solution of Yu Bingleng, is removed
It is shredded after meninx and the big blood vessel of brain, after 37 DEG C of digestion 90min of clostridiopetidase A and DNA enzymatic are added, 1000 revs/min of centrifugation 8min,
It discards supernatant, is transferred in 20% BSA, 4 DEG C of centrifugation 20min of 1000g/min discard liquid at the middle and upper levels, by bottom capilary
It is transferred in training liquid, 1000 revs/min of centrifugation 5min, it is outstanding to be made into capilary section with the DMEM culture solution containing 20% fetal calf serum
Liquid is inoculated in and is covered in 24 hole transwell of Collagen type-I in advance, transwell is moved into carbon dioxide incubator, 37
DEG C, 5%CO2And cultivated under the conditions of saturated humidity change into for 24 hours the endothelium special culture solution containing puromycin continue cultivate 72h,
Change the endothelium special culture solution culture 72h containing Porcine HGF into again, measuring resistance is more than 200 Ω cm2, i.e., in vitro
BBB model is successfully established;
Preparing C6 concentration with the DMEM culture solution containing 10%FBS is 50ng/mL'sDWVAP1-Micelle/C6、DWSW-
Micelle/C6、DCulture solution in transwell upper chamber is sucked out VAP-Micelle/C6 and Micelle/C6 solution, respectively
Above-mentioned solution is added, removes room culture solution respectively at 30min, 1,2 and 4h and surveys its fluorescence intensity, each polymer micelle BBB transhipment
As a result as shown in Figure 9 A;
3)DWVAP1-Micelle is investigated across external bloodtumor barrier ability and is tested
Huve cell HUVEC and U87 is laid on to the upper and lower room of transwell respectively according to the ratio of 1:5, is trained
72h is supported, preparing fluorescence intensity with the DMEM culture solution containing 10%FBS is 50ng/mL'sDWVAP1-Micelle/C6、DWSW-
Micelle/C6、DCulture solution in transwell upper chamber is sucked out VAP-Micelle/C6 and Micelle/C6 solution, respectively
Above-mentioned solution is added, removes room culture solution respectively at 30min, 1,2 and 4h and surveys its fluorescence intensity, each polymer micelle BTB transhipment
As a result as shown in Figure 9 B;
4、DWVAP1-Micelle is in vitro across blood brain barrier or bloodtumor barrier targeting brain tumor verifying
2% low molecule agarose solution is added while hot in 48 orifice plates, every 150 μ L of hole, after being placed at room temperature for cooled and solidified,
Every hole is inoculated with 400 μ L U87 cell suspensions, and cell density is 2 × 103A/hole, is placed in carbon dioxide incubator, 37 DEG C, 5%
CO2And cultivated under the conditions of saturated humidity 7 days and form tumour ball, U87 tumour ball is transferred under the transwell of BBB model
Room is cultivated up to BBB/U87 tumour ball co-culture model, and U87 tumour ball is transferred to room under the transwell of BTB model and is cultivated
Up to BTB/U87 tumour ball co-culture model;
DWVAP1-Micelle/C6、DWSW-Micelle/C6、DVAP-Micelle/C6 and Micelle/C6 solution with
After 50% serum is incubated for 4h, it is 50ng/ that the micella for being incubated for front and back, which is prepared fluorescence intensity with the DMEM culture solution containing 10%FBS,
ML'sDWVAP1-Micelle/C6、DWSW-Micelle/C6、DVAP-Micelle/C6 and Micelle/C6 solution, will
The culture solution of transwell upper chamber is sucked out, and is separately added into above-mentioned solution, continues to cultivate and removes the tumour ball of room after 4h and be placed in fluorescence
Microscopically observation, as a result as shown in Figure 9 C.
Embodiment 7DTargeting verifying in the polymer micelle body of WVAP1 modification
1) preparation carries Micelle/DiR and Micelle/DiD
The preparation method for carrying Micelle/DiR and Micelle/DiD is prepared with Micelle/C6 is carried;
2)DTargeting verifies qualitative experiment in WVAP1-Micelle/DiR body
The Micelle/DiR of the original position U87 Brain Glioma Model mouse difference 100 μ L of tail vein injection,DWVAP1-Micelle/
DiR、DWSW-Micelle/DiR andDVAP-Micelle/DiR anaesthetizes nude mice in injection afterwards for 24 hours, records DiR with living imaging instrument
Fluorescence is in the intracorporal distribution situation of nude mice and carries out fluorescence sxemiquantitative calculating.Then tumour is cut to take pictures and carry out fluorescence sxemiquantitative
(as shown in Fig. 8 C and 8D);
3)DThe distribution of WVAP1-Micelle/DiD normal rat intracerebral
The Micelle/DiD of ICR mouse difference 100 μ L of tail vein injection,DWVAP1-Micelle/DiD、DWSW-
Micelle/DiD andDVAP-Micelle/DiD puts to death mouse in injection 2h and for 24 hours respectively afterwards, it is even then to cut brain tissue
It starches and measures fluorescence (as illustrated in fig. 8e);
4)DWVAP1-Micelle/DiD lotus knurl mouse intracerebral is distributed quantitative experiment
The Micelle/DiD of the original position U87 Brain Glioma Model mouse difference 100 μ L of tail vein injection,DWVAP1-Micelle/
DiD、DWSW-Micelle/DiD andDVAP-Micelle/DiD, respectively inject 2h and for 24 hours when put to death nude mice, then cut packet
Brain tissue containing tumour is homogenized and measures fluorescence (as shown in Figure 8 F).
Embodiment 8 carries the Pharmacodynamics in vitro test of taxol or parithenolide polymer micelle
1) preparation and characterization of taxol or parithenolide polymer micelle are carried
Weigh 1mgDWVAP1-PEG-PLA, 9mg mPEG-PLA and 2mg PTX (taxol), are dissolved in 2mL acetonitrile,
37 DEG C of water-baths, decompression (~0.085MPa) are evaporated, and are formed a film, and room temperature in vacuo is dried overnight, and 2mL physiological saline aquation, CL-4B is added
Column chromatography removes free drug, is made and contains taxolDWVAP1 modification polymer micelle (DWVAP1-Micelle/PTX)。
Carry parithenolide (PTL)DWVAP1 modification polymer micelle (DWVAP1-Micelle/PTL preparation method) is same as above;Swash
Light particle size analyzer determination partial size and PDI (as shown in Figure 10);2,DWVAP1-Micelle/PTX andDWVAP1-Micelle/PTL is external
Pharmacodynamic test
With 4.0 × 103In 96 orifice plates, culture solution is sucked out afterwards for 24 hours for U87 cell inoculation in a/hole, adds 200 μ L a series of
ConcentrationDWVAP1-Micelle/PTX、DWSW-Micelle/PTX、DVAP-Micelle/PTX, Micelle/PTX and Taxol
(taxol) after cultivating 72h, is added MTT solution and continues to cultivate 4h, discard culture solution, 150 μ L DMSO, oscillation to purple is added
Grain dissolution, measure absorbance value 590nm at microplate reader, using mtt assay measure cell survival rate, calculating cell survival rate with
Half lethal dose,DThe test of WVAP1-Micelle/PTL pharmacy in vitro is same as above, as a result as shown in figure 11;
3) the inhibition test that new vessels are formed
It takes the every hole of 24 well culture plates that 50 μ L matrigels are added, is laid in 24 orifice plates, 30min is incubated in 37 DEG C of incubators and is waited for
It is solidified.0.25% pancreatin digests HUVEC cell, with containingDWVAP1-Micelle/PTX、DWVAP1-Micelle/PTL, PTL and
The DMEM culture solution of Taxol is made into single cell suspension, with every hole 1 × 105A cell inoculation is in 24 well culture plates, 37 DEG C, 5%
CO2And 12h is cultivated under the conditions of saturated humidity, observation capillary structure forms (as illustrated in fig. 12);
4) the inhibition test of mimicry vascularization
It takes the every hole of 24 well culture plates that 50 μ L matrigels are added, is laid in 24 orifice plates, 30min is incubated in 37 DEG C of incubators and is waited for
It is solidified.0.25% pancreatin digests U87 cell, with containingDWVAP1-Micelle/PTX、DWVAP1-Micelle/PTL, PTL and
The DMEM culture solution of Taxol is made into single cell suspension, with every hole 1 × 105A cell inoculation is in 24 well culture plates, 37 DEG C, 5%
CO2And 12h is cultivated under the conditions of saturated humidity, observation capillary structure forms (as shown in Figure 12 B).
Embodiment 9 carries the internal pharmacodynamics test of taxol or parithenolide polymer micelle
1)DPharmacodynamics test in WVAP1-Micelle/PTX body
Lotus original position Brain Glioma Model nude mice tail vein distinguish injecting normal saline, Taxol, Micelle/PTX,DWVAP1-Micelle/PTX、DWSW-Micelle/PTX andDVAP-Micelle/PTX.It is 24mg/kg that accumulated dose, which is administered, in PTX,
It is administered within the 6th, 9,12 and 15 day after tumor planting respectively, records the life span of nude mice, Figure 13 shows nude mice life span
Curve, compared with other each groups,DWVAP1-Micelle/PTX significantly extends lotus original position Brain Glioma Model nude mice life span;
2)DPharmacodynamics test in WVAP1-Micelle/PTL body
Lotus original position Brain Glioma Model nude mice tail vein distinguish injecting normal saline, PTL, Micelle/PTL andDWVAP1-
Micelle/PTL.It is 25mg/kg that accumulated dose, which is administered, in PTL, respectively the 6th after tumor planting, administration in 9,12,15 and 17 days, record
The life span of nude mice, nude mice time-survivor curve is as shown in figure 14, compared with other each groups,DWVAP1-Micelle/PTL is aobvious
It writes and extends lotus original position Brain Glioma Model nude mice life span.
The internal pharmacodynamics test of 10 drug-carrying polymer micelle drug combination of embodiment
1)DWVAP1-Micelle/PTL withDThe internal pharmacodynamics test that WVAP1-Micelle/PTX is administered in combination
Lotus original position Brain Glioma Model nude mice tail vein difference injecting normal saline,DWVAP1-Micelle/PTX、DWVAP1-Micelle/PTL、DWVAP1-Micelle/PTX withDWVAP1-Micelle/PTL is administered in combination, and the administration of PTX is total
Dosage is 30mg/kg, and the administration accumulated dose of PTL is 25mg/kg, respectively the 6th after tumor planting, administration in 9,12,15 and 17 days,
The life span of nude mice is recorded, nude mice time-survivor curve is as shown in figure 15, and compared with other each groups, administering drug combinations can be significant
Extend lotus original position Brain Glioma Model nude mice life span;
2)DThe internal pharmacodynamics test that WVAP1-Micelle/PTL and TMZ is administered in combination
Lotus original position Brain Glioma Model nude mice tail vein difference injecting normal saline,DWVAP1-Micelle/PTL takes orally
Stomach-filling TMZ and TMZ withDWVAP1-Micelle/PTL is administered in combination, and the administration accumulated dose of PTL is 25mg/kg, the administration of TMZ
Accumulated dose is 50mg/kg, respectively the 6th after tumor planting, administration in 9,12,15 and 17 days, records the life span of nude mice, nude mice
Time-survivor curve is as shown in figure 16, and compared with other each groups, administering drug combinations can significantly extend lotus original position Brain Glioma Model
Nude mice life span.
Tumor tissue section after the detection administration of embodiment 11
1) tumor vessel inhibits test
Tumor bearing nude mice is put to death taking-up brain tissue and is fixed, specimens paraffin embedding slices carry out CD31 immunohistochemistry after the completion of administration
Dyeing and the bis- dyes of PAS, are observed continuously 3 high power fields under ordinary optical microscope, count CD31 positive vessels number, as a result such as
Shown in Figure 17;
2) tumor death is tested
Tumor bearing nude mice is put to death taking-up brain tissue and is fixed, make frozen section, examined by TUNEL method after the completion of administration
Tumor death situation is surveyed, the dUTP Nick End labelling method (Terminal mediated using terminal deoxynucleotidyl transferase (TDT)
Deoxynucleotidyl Transferase-mediated dUTP nick end labeling, TUNEL) detection tumour it is thin
The apoptosis degree of born of the same parents, comprising steps of paraffin section routinely dewaxes to water;PBS is rinsed 3 times, each 3min;0.3%H2O2Solution room
Temperature processing 20min;20 37 DEG C of μ g/mL Proteinase K digestion 20min;PBS is rinsed 3 times, each 3min;Every slice is added dropwise
30 μ L of TUNEL mixed liquor (TDT and biotin-dNTP) is placed in 37 DEG C of incubation 60min in wet box;Positive findings are nucleus in palm fibre
Yellow or sepia, the brown particle positive is determined as apoptotic cell in nucleus;5 are observed continuously under ordinary optical microscope
A high power field counts positive cell number, and percentage shared by positive cell number is apoptotic index (such as Figure 18 in the inner cell of the visual field
It is shown);
3) tumor stem cell Inhibition test
For tumor bearing nude mice after the completion of administration, conventional treatment takes brain tissue to be fixed, and makees frozen section, is contaminated by CD133
Color method detects the number of brain Tumor Stem Cells, and positive findings are that nucleus is in brown color or sepia, brown particle in nucleus
The positive is determined as tumor stem cell cell, and 5 high power fields are observed continuously under ordinary optical microscope, counts positive cell
Number, as a result as shown in figure 19.
Claims (19)
1. a kind of overall process target polypeptide WVAP, which is characterized in that the overall process target polypeptide WVAP is targeted in brain capillary
Chrotoplast simultaneously crosses over blood brain barrier, target tumor neovascular endothelium cell and crosses over bloodtumor barrier, while target tumor
WSW is covalently attached with VAP by bridge crosslinking structure (linker) and is formed by mimicry blood vessel, tumour cell and tumor stem cell
(LWVAP:PAVRTNS-linker-SYPGWSW (LWVAP1)、SNTRVAP-linker-SYPGWSW(LWVAP2)、SYPGWSW-
linker-PAVRTNS(LWVAP3) and SYPGWSW-linker-SNTRVAP (LWVAP4);DWVAP:DSDNDTDRDVDADP-
linker-DWDSDWDGDPDYDS(DWVAP1)、DPDADVDRDTDNDS-linker-DWDSDWDGDPDYDS(DWVAP2)、DWDSDWDGDPDYDS-linker-DSDNDTDRDVDADP(DWVAP3) andDWDSDWDGDPDYDS-linker-DPDADVDRDTDNDS
(DWVAP4);LDWVAP:PAVRTNS-linker-DWDSDWDGDPDYDS(LDWVAP1)、SNTRVAP-linker-DWDSDWDGDPDYDS(LDWVAP2)、DWDSDWDGDPDYDS-linker-PAVRTNS(LDWVAP3)、DWDSDWDGDPDYDS-linker-
SNTRVAP(LDWVAP4)、DSDNDTDRDVDADP-linker-SYPGWSW(LDWVAP5)、DPDADVDRDTDNDS-linker-
SYPGWSW(LDWVAP6)、SYPGWSW-linker-DSDNDTDRDVDADP(LD) and SYPGWSW-linker- WVAP7DPDADVDRDTDNDS(LDWVAP8))。
2. overall process target polypeptide WVAP according to claim 1, characterized in that the bridge crosslinking structure is amino fatty acid
And its derivative, amino aromatic acid and its derivative or oligopeptides.
3. overall process target polypeptide WVAP according to claim 1, characterized in that the overall process target polypeptide WVAP is for being situated between
The leap blood brain barrier and bloodtumor barrier of drug molecule or nanoscale medicine delivery system are led, target tumor new vessels, tumour are quasi-
State blood vessel, tumour cell and tumor stem cell realize that drug molecule or nano medicament carrying system turn to brain, brain tumor or with brain
Move the targeted delivery of feature periphery tumour.
4. overall process target polypeptide WVAP according to claim 1, characterized in that the overall process target polypeptide WVAP is used for
The medicinal composition of its modification, polymer carrier are constructed by introducing active function groups in molecule, introduces the position of functional group
Point is nitrogen end, carbon teminal or the bridge crosslinking structure of WVAP.
5. overall process target polypeptide WVAP according to claim 1, characterized in that introduce after sulfydryl and contain maleimide
WVAP-X compound is made in the image substance reaction of group.
6. overall process target polypeptide WVAP as described in claim 5, characterized in that in the WVAP-X compound, X is glimmering
Stimulative substance Fluorescein, nir dye Cy7, IR820, DiR, nuclear magnetic resonance image agent Gd-DTPA, irradiation image agent99mTc-
DTPA。
7. overall process target polypeptide WVAP according to claim 1, characterized in that sensitive by the hydrazone bond of pH sensitivity, pH
Boric acid rouge key, disulfide bond and therapeutic agent connect, or are directly condensed with polypeptide drugs, and WVAP-Y compound is made.
8. overall process target polypeptide according to claim 7, characterized in that in the WVAP-Y compound, Y is antitumor
Adriamycin and Epi-ADM class anthracene nucleus medicament, taxol and Docetaxel and Cabazitaxel taxanes medicine in drug
Object, camptothecine and hydroxycamptothecin and Irinotecan camptothecine, vincristine and vinorelbine vinca alkaloids drug,
Bortezomib helps rice class drug, p53 activates peptide medicament.
9. overall process target polypeptide WVAP according to claim 1, characterized in that introduce sulfydryl after with maleimation
The connection of polyethylene glycol-Z compound, is made WVAP- polyethylene glycol-Z compound.
10. pressing claim 9 overall process target polypeptide WVAP, in the WVAP- polyethylene glycol-Z compound, Z is phosphatide, gathers
Lactic acid (PLA), lactic-co-glycolic acid (PLGA), polycaprolactone (PCL).
11. overall process target polypeptide WVAP according to claim 1, characterized in that the WVAP- polyethylene glycol-phospholipids
Compound is used to prepare liposome delivery systems, polymer micelle delivery system and polymer disc delivery system.
12. overall process target polypeptide WVAP as described in claim 10, characterized in that the WVAP- polyethylene glycol cream
Sour compound, WVAP- polyethylene glycol-lactic-co-glycolic acid compound or WVAP- polyethylene glycol-polycaprolactone compound
It is used to prepare polymer micelle delivery system or nanoparticle delivery system.
13. by overall process target polypeptide WVAP described in claim 11,12, characterized in that the liposome delivery systems,
Polymer micelle delivery system, polymer disc delivery system or nanoparticle delivery system are for containing diagnostic medicine.
14. overall process target polypeptide WVAP according to claim 13, characterized in that the delivery system package-contained diagnosis
Drug is fluorescent material coumarin 6, FAM, nir dye Cy7, IR820, DiR, DiD, nuclear magnetic resonance image agent Gd-DTPA.
15. by overall process target polypeptide WVAP described in claim 11,12, characterized in that the liposome delivery systems,
Polymer micelle delivery system, polymer disc delivery system or nanoparticle delivery system are for containing tumor therapeutic agent.
16. overall process target polypeptide WVAP according to claim 15, characterized in that the delivery system package-contained tumour
Therapeutic agent is adriamycin and Epi-ADM anthracene nucleus medicament, taxol and Docetaxel and Cabazitaxel taxanes medicine
Object, camptothecine and hydroxycamptothecin and Irinotecan camptothecine, vincristine and vinorelbine vinca alkaloids drug,
Bortezomib and Carfilzomib proteasome inhibitor, parithenolide lactone drug, p53 activating peptide and melittin, scorpion venom
Peptide and antibacterial peptide polypeptide drug.
17. overall process target polypeptide WVAP according to claim 15, characterized in that the delivery system package-contained tumour
Therapeutic agent and other tumor therapeutic agent drug combinations or delivery system contain different tumor therapeutic agent drug combinations and use
In preparation synergistic treatment brain tumor or synergism medicine with brain metastes feature periphery tumour.
18. overall process target polypeptide WVAP according to claim 17, characterized in that the tumor therapeutic agent contained is
Adriamycin, Epi-ADM, taxol, Docetaxel, Cabazitaxel, camptothecine, hydroxycamptothecin, 9-nitrocamptothecin, she
It is vertical to replace health, vincristine, vinorelbine, bortezomib, Carfilzomib, parithenolide, p53 activating peptide, melittin, scorpion venom
Peptide, antibacterial peptide, it is described associated with other tumor therapeutic agents be selected from Temozolomide, cyclophosphamide, Etoposide, mercaptopurine, Ji
His shore of west, cytarabine, 5 FU 5 fluorouracil, Teniposide, Epothilones, actinomycin D, mitoxantrone, mitomycin, it is rich come
Mycin replaces Buddhist nun's class drug, platinum medicine, bevacizumab or trastuzumab.
19. overall process target polypeptide WVAP according to claim 17, characterized in that the drug combination wherein, by wrapping
Carry the adriamycin for inhibiting growth of tumour cell, Epi-ADM, taxol, Docetaxel, Cabazitaxel, camptothecine, hydroxyl happiness
Tree alkali, 9-nitrocamptothecin, Irinotecan, vincristine, vinorelbine tumor therapeutic agent delivery system have with containing
The bortezomib of resisting tumour stem cells effect, Carfilzomib, parithenolide tumor therapeutic agent delivery system drug combination,
Or with contain polypeptide p53 activating peptide, melittin and scorpion venom peptide with antitumor action, antibacterial peptide tumor therapeutic agent passs medicine
System combined medication.
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111004307A (en) * | 2019-12-24 | 2020-04-14 | 中山大学 | Indocyanine green compound for treating early brain glioma and preparation method and application thereof |
CN112641953A (en) * | 2019-10-10 | 2021-04-13 | 复旦大学 | Targeting functional molecule modified antibody compound |
CN113171468A (en) * | 2020-01-23 | 2021-07-27 | 复旦大学 | Whole-process targeting molecule and application thereof in construction of drug delivery system |
CN113372412A (en) * | 2021-04-09 | 2021-09-10 | 上海交通大学医学院附属第九人民医院 | Cell-targeted polypeptide for treating bone tumor and preparation method and application thereof |
CN116869968A (en) * | 2023-09-07 | 2023-10-13 | 四川大学 | Nanoparticulate targeting brain and brain glioma, and synthesis method and application thereof |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103012562A (en) * | 2011-09-24 | 2013-04-03 | 复旦大学 | Dual-targeting D-configuration polypeptides and drug delivery system thereof |
CN103417480A (en) * | 2013-07-11 | 2013-12-04 | 四川大学 | Novel polypeptide modified tumor targeted liposome of targeted integrin receptor |
CN103951754A (en) * | 2014-03-26 | 2014-07-30 | 中国药科大学 | Anti-tumor bispecific miniaturized antibody with double functions of targeting therapy and detection |
CN104497147A (en) * | 2014-12-23 | 2015-04-08 | 四川大学 | Double receptor identified serial cell-penetrating peptide modified tumor targeted nano drug delivery system |
CN104558117A (en) * | 2013-10-25 | 2015-04-29 | 复旦大学 | Acetylcholine receptor-mediated targeting D-configuration polypeptide and application thereof |
CN105050612A (en) * | 2012-10-19 | 2015-11-11 | 维克特-霍鲁斯公司 | Compositions and methods for drug delivery |
CN105294831A (en) * | 2015-10-10 | 2016-02-03 | 四川大学 | Tumor-targeting novel polypeptide |
CN106333926A (en) * | 2015-07-10 | 2017-01-18 | 复旦大学 | Brain tumor multiple targeting drug delivery system of stability polypeptide mediated cross-barrier film |
-
2017
- 2017-08-11 CN CN201710684008.8A patent/CN109384850A/en active Pending
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103012562A (en) * | 2011-09-24 | 2013-04-03 | 复旦大学 | Dual-targeting D-configuration polypeptides and drug delivery system thereof |
CN105050612A (en) * | 2012-10-19 | 2015-11-11 | 维克特-霍鲁斯公司 | Compositions and methods for drug delivery |
CN103417480A (en) * | 2013-07-11 | 2013-12-04 | 四川大学 | Novel polypeptide modified tumor targeted liposome of targeted integrin receptor |
CN104558117A (en) * | 2013-10-25 | 2015-04-29 | 复旦大学 | Acetylcholine receptor-mediated targeting D-configuration polypeptide and application thereof |
CN103951754A (en) * | 2014-03-26 | 2014-07-30 | 中国药科大学 | Anti-tumor bispecific miniaturized antibody with double functions of targeting therapy and detection |
CN104497147A (en) * | 2014-12-23 | 2015-04-08 | 四川大学 | Double receptor identified serial cell-penetrating peptide modified tumor targeted nano drug delivery system |
CN106333926A (en) * | 2015-07-10 | 2017-01-18 | 复旦大学 | Brain tumor multiple targeting drug delivery system of stability polypeptide mediated cross-barrier film |
CN105294831A (en) * | 2015-10-10 | 2016-02-03 | 四川大学 | Tumor-targeting novel polypeptide |
Non-Patent Citations (6)
Title |
---|
DANNI RAN 等: "D-Retroenantiomer of Quorum-Sensing Peptide-Modified Polymeric Micelles for Brain Tumor-Targeted Drug Delivery", 《ACS APPLIED MATERIALS & INTERFACES》 * |
EVELIEN WYNENDAELE等: "Quorum Sensing Peptides Selectively Penetrate the Blood-Brain Barrier", 《BBB-PENETRATION OF QUORUM SENSING PEPTIDES》 * |
FORTUNATO FERRARAA等: "Targeted molecular一genetic imaging and ligand-directed therapy in aggressive variant prostate cancer", 《PNAS》 * |
JAMI MANDELIN等: "Selection and identification of ligand peptides targeting a model of castrate-resistant osteogenic prostate cancer and their receptors", 《PNAS》 * |
MIN LIU等: "D-Peptides as Recognition Molecules and Therapeutic Agents", 《THE CHEMICAL RECORD》 * |
ZAKIA BELHADJ 等: "Design of Y-shaped targeting material for liposome-based multifunctional glioblastoma-targeted drug delivery", 《JOURNAL OF CONTROLLED RELEASE》 * |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112641953A (en) * | 2019-10-10 | 2021-04-13 | 复旦大学 | Targeting functional molecule modified antibody compound |
CN111004307A (en) * | 2019-12-24 | 2020-04-14 | 中山大学 | Indocyanine green compound for treating early brain glioma and preparation method and application thereof |
CN111004307B (en) * | 2019-12-24 | 2022-09-13 | 中山大学 | Indocyanine green compound for treating early brain glioma as well as preparation method and application thereof |
CN113171468A (en) * | 2020-01-23 | 2021-07-27 | 复旦大学 | Whole-process targeting molecule and application thereof in construction of drug delivery system |
WO2021147917A1 (en) * | 2020-01-23 | 2021-07-29 | 复旦大学 | Application of systematic targeting molecule and drug complex thereof in constructing drug delivery system |
CN114981310A (en) * | 2020-01-23 | 2022-08-30 | 复旦大学 | Whole-process targeting molecule and application of drug compound thereof in construction of drug delivery system |
CN113372412A (en) * | 2021-04-09 | 2021-09-10 | 上海交通大学医学院附属第九人民医院 | Cell-targeted polypeptide for treating bone tumor and preparation method and application thereof |
CN116869968A (en) * | 2023-09-07 | 2023-10-13 | 四川大学 | Nanoparticulate targeting brain and brain glioma, and synthesis method and application thereof |
CN116869968B (en) * | 2023-09-07 | 2023-11-24 | 四川大学 | Nanoparticulate targeting brain and brain glioma, and synthesis method and application thereof |
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