CN102875647A - Ligand polypeptide and medicine transferring system combined with specificity of CD40L protein - Google Patents
Ligand polypeptide and medicine transferring system combined with specificity of CD40L protein Download PDFInfo
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Abstract
The invention discloses ligand polypeptide and a medicine transferring system combined with the specificity of CD40L protein. The ligand polypeptide combined with the specificity of the CD40L protein comprises an amino acid sequence showed in SEQ ID NO.1. The medicine transferring system provided by the invention comprises the ligand polypeptide, medicine carrying system and at least one kind of active substance, wherein the medicine carrying system is specifically liposome; the at least one kind of active substance is the substance which is required to be transferred to specified positions in the body; the ligand polypeptide provided by the invention has excellent targeting functions for CD40L; aiming at immune diseases in the body with high expression of CD40L, a reagent kit used for early diagnosis and therapeutic medicine with specificity can be developed; and meanwhile, the polypeptide can combined with the liposome to be used as targeting medicine carrier, so that the medicine transferring system is developed into a targeting medicine transferring system which is aiming at immune diseases in the body.
Description
Technical field
The present invention relates to the protein and peptide technical field, relate in particular to a kind of ligand polypeptide and delivery system of being combined with the CD40L protein-specific.
Background technology
CD40L is tumour necrosis factor (TNF) superfamily member, is II type membranin, mainly expresses at the CD4+T cell surface, comprises Th0, Th1 and Th2 cell subsets.CD40L also expresses at other lymphocytic cell surfaces, such as CD8+T cell or the B cell surface of activation.Studies show that functional CD40L also expresses non-lymphocyte in hematopoiesis system, such as the surface of Activated platelet cell.The T cell of activation can reach the peak at transient expression CD40L in rear tens minutes of the activation at 6 hours, expression amount begins to descend subsequently.
CD40/CD40L is a pair of important costimulatory molecules in the specific immune response system, participates in humoral immunization and the cell immune response of body.This signal path can raise activating T cell surface costimulatory molecules B7(CD80/CD86) family member's expression; Increase other cell surfaces CD23, CD54, the expression of CD95 and LT2 β; The isotype conversion of induction of immunity sphaeroprotein IgG, IgA and IgE; Avoid the B cell because Fas(CD95) or the crosslinked apoptosis that causes of IgM of antigen induction; Induce the feminine gender screening of B cell etc.
The CD40/CD40L signal path is relevant with a lot of autoimmune disorders, and it can cause body to produce the immunopathogenesis reaction unusually.Lymphocyte CD 40L expression amount calibration ordinary person level in rheumatoid arthritis (RA) the patient synovial fluid increases.In multiple sclerosis (MS), detect equally the situation of CD40L high expression level.Compared to the normal people, MS patient blood medium size lymphocyte CD40L positive T cell rate significantly increases, and the expression level of the T cell of former expression CD40L also increases to some extent.Because to pass to path relevant with disease of immune system for the CD40/CD40L signal, quantity research is finished treatment to disease for blocking this signal path greatly.In the CIA Research of Animal Model for Study corresponding with RA, anti-CD 40 L monoclonal antibody CIA capable of blocking occurs, and comprises that suppressing antigen presenting cell produces IL-12, suppresses IgG secretion and local joint scavenger cell and produces NO, reduces the joint part inflammatory cell infiltration.In addition in the research of the EAE of corresponding MS mouse model, the monoclonal antibody of anti-CD 40 L can not only occur at the early stage EAE of prevention of morbidity, comprises the secretion that suppresses IL-12, stops the activation of CD4+Th1 cell, the expression of downward modulation ICAM adhesion molecule reduces monocyte and produces NO etc.In follow-up relevant experimentation on animals and clinical experiment, find mechanism that anti-CD40L antibodies plays a role except blocking-up CD40/CD40L signal path, comprise also that outward the antibody Fc section mediation removes the CD40L+T cell of activation.
Enter several antibody of clinical experiment, all suspended clinical trial because of the situation that thrombus occurred.The formation of this thrombus may be because the FcRIIA receptors bind on the thrombocyte in the Fc section on the anti-CD40L antibodies and the blood, causes crosslinked between the thrombocyte, causes the blood aggegation embolism to occur.The improvement that follow-up on this basis antagonist carries out, removal can cause the Fc section of thrombus, only keep the Fab section on the antibody, still can bring into play the effect of its blocking-up CD40/CD40L signal path.But simultaneously because of the removal of Fc section, just can't reach the effect of CD40L+T cell clearance.
Defective and limitation for the antibody drug of blocking the CD40/CD40L signal path, can further transform its Research Thinking: 1. screening is for the ligand polypeptide of CD40L, this polypeptide is used as the Fab section of antibody, reach the effect of blocking-up CD40/CD40L signal path.2. in conjunction with target ligand polypeptide and the liposome drug delivery technologies of CD40L, by the cell of polypeptide target to high expression level CD40L, release targetedly cell toxicity medicament, reach the effect that suppresses CD40L+T cell proliferation.Existing trimerization polypeptide for CD40 occurs at present, and on cell levels, effectively suppressed the CD40/CD40L signal path, but experiment report in the further body is not arranged, do not have for the ligand polypeptide of CD40L in addition yet and realize blocking the report that the CD40/CD40L signal is gone the same way.
Liposome be with the double-deck phospholipid molecule similar of microbial film the artificial rust structure, the inner imitated vesicle structure that forms sealing, can coated water-soluble medicine, in lipid bilayer, also can be written into fat-soluble medicine simultaneously.Except can medicine carrying, liposome also has good biocompatibility, and the modification through the contour hydrophilic pair of property molecule of DSPE-PEG2000 prolongs its cycling time in blood, increases drug release also action time.With targeted molecular, such as polypeptide, monoclonal antibody etc. are connected on the liposome simultaneously, make and have the initiatively liposome system of targeting, can identify target cell and be combined with the acceptor molecule of surface of cell membrane, be conducive to the liposome internalization, discharge in cell with the promotion medicine, thereby improve drug effect.
Thus, the CD40L target polypeptide is combined with the liposome medicine carrying, realizes the blocking-up of CD40/CD40L signal path and kill and wound the effect of high expression level CD40L+T cell.Can reach like this result for the treatment of same with CD40L antibody, but the blood coagulation side effect that can avoid antibody to bring.
Summary of the invention
The object of the present invention is to provide a kind of ligand polypeptide and the delivery system that can be combined with the CD40L protein-specific.This ligand polypeptide is connected with distearyl glyceryl phosphatide thanomin polyoxyethylene glycol maleimide, make the target synthetic materials, then use the target synthetic materials further to prepare target liposomes, measure target synthetic materials and target liposomes to the impact of inside and outside cell and CD40L high expression level disease model.
The objective of the invention is to be achieved through the following technical solutions:
First aspect the present invention relates to a kind of ligand polypeptide of being combined with the CD40L protein-specific, comprises the aminoacid sequence shown in the SEQ IDNO.1.
Preferably, described ligand polypeptide contains more than or equal to two aminoacid sequences shown in SEQ ID NO.1.
Preferably, described ligand polypeptide comprises sequence shown in the SEQ ID NO.1, and adds linking group or connection aminoacid sequence at its C-terminal and/or N-terminal.
Second aspect the present invention relates to sequestered polypeptide, pattern of fusion polypeptide, chimeric class polypeptide or the polymkeric substance take ligand polypeptide as monomer that a kind of aforesaid ligand polypeptide of being combined with the CD40L protein-specific forms.
The third aspect the present invention relates to a kind of delivery system, and this system comprises: aforesaid ligand polypeptide of being combined with the CD40L protein-specific, drug-loading system, at least a active substance.
Preferably, described ligand polypeptide can be connected to following arbitrary body structure surface: nanoparticle, polymkeric substance, liposome, vesica, solid lipid particulate, micella, carbon nanotube, organoid, lipoprotein.
Preferably, described drug-loading system is specially liposome, its moiety comprises: phosphatidylcholine, phosphatidylethanolamine, phosphatidyl glycerol, phosphatidylinositols, phosphatidylserine, phosphatidic acid, Val, sphingophospholipid, cerebroside, Sphingolipids,sialo, ergosterol, cholesterol, cation lipid, lysophospholipid, polyethyleneglycol modified lipid, the lipid that ligand polypeptide is modified.
Preferably, described at least a active substance is: antitumor drug, antimetabolite, spindle body toxic alkaloid, cytotoxicity/antitumor antibiotics, topoisomerase enzyme inhibitor, photosensitizers, kinase inhibitor, microbiotic, antibiotic, anti-inflammatory drug, immunosuppressor, anti-infectives, antiviral, insect repellent, antiparasitic compound is used for ultrasonic contrast, pneumoradiography, the diagnostic reagent of nuclear medicine contrast medium, goal gene, the antisense oncogene, suicide gene, apoptosis gene, cytokine gene, or the combination of said gene, or contain the carrier for expression of eukaryon DNA of said gene.
The present invention has following beneficial effect:
(1) be connected to synthetic materials with distearyl glyceryl phosphatide thanomin polyoxyethylene glycol maleimide behind of the present invention and ligand polypeptide modified tetramino that the CD40L protein-specific the is combined acid connecting joint, the fluorescent lipid body that is prepared into shows at external cell with the CD40L high expression level and well is combined effect; Through the fluorescent substance mark, the basic lymphoglandula with being rich in the T cell of this polypeptide distribution in vivo coincide.
(2) be connected to synthetic materials with distearyl glyceryl phosphatide thanomin polyoxyethylene glycol maleimide behind this ligand polypeptide modified tetramino acid connecting joint, the methotrexate liposome that is prepared into shows stronger lethal effect at external cell to the CD40L positive, has shown better result for the treatment of in the EAE animal model; The polyethyene diamine that this ligand polypeptide is modified shows better transfection effect to the cell of CD40L high expression level.
(3) ligand polypeptide of being combined with the CD40L protein-specific of the present invention not only has the advantage of traditional antibody molecule, and molecular weight is little, tissue penetration is strong, has good CD40L targeting, autoimmune disorder for number of C D40L high expression level can be developed as early diagnosis kit and specific medicine, be combined with liposome can also be as target medicine carrier for this polypeptide simultaneously, develop into the targeted drug delivery system for autoimmune disorder, realize stronger result for the treatment of.
Description of drawings
Fig. 1 is the preparation flow figure of CD40L target polypeptide;
Fig. 2 is CD40L target polypeptide and the interior distribution plan of the time dependent body of control peptide thereof of Cy5.5 mark;
Fig. 3 contains the fluorescent lipid body of 2% polypeptide lipid in conjunction with the fluorescence intensity synoptic diagram of Jurkat cell strain before and after the CD40L up-regulated;
Fig. 4 contains the fluorescence intensity synoptic diagram that the fluorescent lipid body of 2% polypeptide lipid is combined with rheumatoid arthritis person joint hydrops medium size lymphocyte;
Fig. 5 is fluorescent lipid body and the fluorescence intensity synoptic diagram of mouse spleen lymphocyte before and after the CD40L up-regulated that contains 2% polypeptide lipid;
Fig. 6 contains the fluorescence intensity synoptic diagram that the fluorescent lipid body of different ratios polypeptide lipid is combined with the EAE mouse spleen lymphocyte;
Fig. 7 is that the cytotoxicity to the Jurkat cell strain of the molecular targeted peptide modified methotrexate liposome of CD40L is investigated the situation synoptic diagram;
Fig. 8 is that the EAE mouse is used the molecular targeted peptide modified methotrexate liposome therapeutic clinical score changing conditions synoptic diagram of CD40L;
Fig. 9 is that the EAE mouse uses behind the molecular targeted peptide modified methotrexate liposome therapeutic of CD40L that the CD40L positive T cell forms the changing conditions synoptic diagram in the peripheral blood.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are not used in for explanation the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, for example the Sambrook equimolecular is cloned: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The present invention relates to a kind of ligand polypeptide of being combined with the CD40L protein-specific, the applicant has investigated simultaneously the binding ability of this peptide modified liposome and CD40L high expressing cell and has carried out external evaluation, prepare fluorescently-labeled polypeptide it is carried out the investigation that distributes in the body, prove conclusively the ability of this polypeptide and CD40L high expressing cell specific binding.The process that the CD40L molecular specificity target polypeptide that the present invention relates to (ligand polypeptide of namely being combined with the CD40L protein-specific) obtains is as shown in Figure 1:
One, computer-aided screening CD40L target ligand candidate polypeptide
Use the Shanghai dawn A4000 of computer center to calculate the unit computing, make up the protein binding model based on CD40/CD40LX ray diffraction crystalline structure, make up and the incorporative peptide library of CD40L, the polypeptide ligand molecule of screening target CD40L.
1) uses SPDBV3.7, manually produce the CD40L dimeric structure, be used in conjunction with screening
2) PSCAN2.2.2 scanning dimeric structure surface produces the pocket that is fit to combination
3) polypeptide fragment with the upper corresponding binding pocket position of CD40 carries out preliminary screening, uses Autodock3.0.5 to utilize Lamarckian genetic algorithm calculations incorporated energy with the docking energy and with its ordering
4) analyze each amino acid of candidate's polypeptide to the hydrogen bond contribution of combination, candidate's polypeptide is carried out point mutation, generate the skewed popularity peptide library that stronger binding ability is arranged with the CD40L dimer, still use Autodock3.0.5 to utilize Lamarckian genetic algorithm to calculate in the storehouse all polypeptide in conjunction with energy and docking energy and with its ordering, obtain CD40L target polypeptide candidate peptide.
Two, the SPR of CD40L candidate polypeptide ligand screening
Use the BIAcore X100 of GE healthcare, the CM5 chip uses the competition combined techniques, by the signal attenuation degree that surface plasma resonance technology produces, screens the polypeptide ligand molecule of target CD40L on the albumen aspect.
1) according to the chip operation instruction, hCD40 is fixed on the CM5 chip
2) add hCD40L in the moving phase, investigate signal response with the combination degree of reflection CD40L and CD40
3) add simultaneously hCD40L and a certain proportion of candidate's polypeptide in the moving phase, investigate signal response
4) according in conjunction with response signal intensity, calculate each candidate's peptide to the competition per-cent of CD40L and ordering, obtain the CD40L target polypeptide.
Three, peptide sequence is synthetic
Candidate's polypeptide and CD40L targeted polypeptide ESEEED(are called for short JD6, shown in SEQ ID NO.1), control peptide DSDDDE(with identical charges number is called for short JD6c) and the ESEEEDGGGC(that contains the halfcystine end of the design for the convenient chemistry of subsequent experimental connects shown in SEQ ID NO.2) and the DSDDDEGGGC polypeptide synthetic by the triumphant prompt biological medicine in Chengdu Development Co., Ltd, all pass through HPLC purifying and Mass Spectrometric Identification, purity is greater than 95%, and molecular weight conforms to theoretical value.
The polypeptide and preparation method thereof of deriving of embodiment 1, CD40L targeted polypeptide
Present embodiment relates to the polypeptide of deriving of described CD40L targeted polypeptide, and its preparation method comprises the steps:
1) get 1mg Cy5.5-NHS and be dissolved in 400 μ l DMSO(methyl-sulphoxides), the 0.8mg polypeptide is dissolved in 400 μ l DMSO;
2) in system, add 15 μ l triethylamines behind equal-volume mixing Cy5.5-NHS and the polypeptide, place the isothermal vibration reactor, room temperature, the lucifuge reaction is spent the night;
3) to place molecular weight cut-off be 1000 dialysis tubing to reaction system, uses lucifuge dialysis under the PBS room temperature, changes water three times, removes DMSO, triethylamine and free Cy5.5-NHS;
4) the CY5.5-polypeptide behind the purifying keeps in Dark Place in-80 degrees centigrade.
Compliance test result detects polypeptide JD6 and contrast polypeptide JD6c distribution situation in vivo thereof.The effect process is as follows: the polypeptide of aforementioned preparation-Cy5.5 solution is entered in the Mice Body by tail vein injection, the amount of every injected in mice Cy5.5 mark is 10ug, injection is rear according to different time points, utilize the distribution situation of small animal living body fluorescence imaging system scan polypeptide in Mice Body, the analysis software that scanning result utilizes system to carry is processed and is analyzed, as shown in Figure 2, as shown in Figure 2: polypeptide is through after the Cy5.5 fluorescent mark, and distribution namely represents polypeptide distribution situation in vivo in the body of fluorescence.Use pseudo-color expression fluorescence intensity among the figure, red fluorescence intensity is the highest, and purple is minimum, improves the readability of picture with this.Use in addition analysis software eXplore Optix OptiView 1.0.0.0, the scanning result of each time point is carried out normalization method, the convenient polypeptide of investigating each position of different time points mouse distributes and aggregation extent.Can see among Fig. 2 polypeptide JD6 in vivo in time prolongation be enriched to gradually the lymphoglandula at upper limbs neck chest place, and in these lymphoid organs are protected, hold more than 6 hours.Polypeptide in contrast, JD6c is in the time of 30 minutes, control peptide also equally appears in some lymphoglandula with JD6, but obviously in follow-up time point, the fluorescence of these gatherings has disappeared, contrast is described, and polypeptide is very fast has been removed by health, does not rest on lymphoglandula, and it is that to have a lymphsystem specific that this explanation polypeptide JD6 distributes in the body of mouse.This result fits like a glove with the situation that is rich in the T cell of JD6 target in the lymphsystem.
Present embodiment relates to a kind of synthetic materials of the CD40L of having targeting, and this synthetic materials is connected with Mal-PEG2000-DSPE by the polypeptide of deriving of CD40L molecular specificity target polypeptide and gets.The sequence of described CD40L molecular specificity target polypeptide is ESEEED, adds GGGC at the C end when synthetic, and then obtains the polypeptide of deriving, and its sequence is ESEEEDGGGC; Sulfydryl free on the halfcystine of this polypeptide of deriving just is connected to DSPE-2000-Maleimide(distearyl glyceryl phosphatide thanomin Macrogol 2000 maleimide with the maleimide base group reaction, polyoxyethylene glycol wherein also can be selected PEG5000) on, the synthetic materials that obtains having the CD40L targeting; Further, described CD40L molecular specificity target polypeptide also can directly be connected with DSPE-2000Maleimide; Obtain the synthetic materials that another kind has the CD40L targeting.
The preparation of the synthetic materials of the described CD40L of having targeting (wherein polypeptide is CD40L molecular specificity target polypeptide) comprises the steps:
1, weighing DSPE-PEG2000-Maleimide lipid adds chloroform to 10mg/ml, dissolves in eggplant-shape bottle, and nitrogen dries up, and forms lipid membrane;
2,1.5 times (molar ratios) are dissolved in HEPES(hydroxyethyl piperazine second thiosulfonic acid buffer salt solution in the polypeptide powder of lipid under nitrogen protection) in, adding has in the eggplant-shape bottle of lipid membrane;
3, in the isothermal reactor, the lower 10 degrees centigrade of reactions of nitrogen protection are spent the night;
4, reaction system is transferred in the dialysis tubing of WMCO1000, uses the deionized water dialysed overnight three times, the synthetic materials that obtains having the CD40L targeting, i.e. polypeptide-PEG-DSPE.
The molecular targeted peptide modified fluorescent lipid body of embodiment 3, CD40L
Present embodiment relates to a kind of fluorescent lipid body, and this fluorescent lipid body is the molecular targeted peptide modified fluorescent lipid body of CD40L, and its preparation method comprises the steps:
1) according to molar ratio EPC(Yelkin TTS): the CHOL(cholesterol): fluorescein DHPE(is fluorescein-labeled 1,2-palmityl phosphatidylethanolamine)=60:40:1,70:30:1 or 80:20:1 take by weighing, fully be dissolved in the trimethyl carbinol;
2) in the lipid t-butanol solution, dropwise add polypeptide-PEG-DSPE aqueous solution (embodiment 2 preparation), keeping system clarifies at the most that the molar ratio of peptide lipid and fluorescein DHPE is respectively 0.1:1,0.5:1,1:1,2:1,3:1,4:1,5:1;
3) mixing polypeptide lipid and other lipid composition, quick freezing in liquid nitrogen places Freeze Drying Equipment-70 degree centigrade freeze-drying;
4) use the rapid hydrated lipidic component of PBS, ultrasonic 30 seconds of lucifuge water-bath, preparation liposome;
5) the lucifuge use is extruded instrument liposome is crossed the 200nm polycarbonate membrane 11 times;
6) use the laser particle analyzer dynamic light scattering method to detect liposome particle diameter and distribution.
Compliance test result:
(1) cell culture experiments
Jurkat is containing 10%FBS(peptide bovine serum), penicillin (100units/ml), Streptomycin sulphate (100mg/ml), Jurkat cell 1640 substratum in cultivate, 37 the degree, 5%CO2.
Jurkat uses 12-o-myristoyl Buddhist ripple acetic ester-13 and ionomycin to stimulate, and raises the expression of its CD40L.Use the TPA of 6ng/ml and the Ionomycine of 1 μ g/ml to stimulate Jurkat, 37 degrees centigrade of 5%CO in containing the RPIM1640 substratum of 20%FBS
2Continue to cultivate 6 hours, detect the expression level of its CD40L and carry out subsequent experimental.
Fig. 3 contains the fluorescent lipid body of 2% polypeptide lipid in conjunction with the fluorescence intensity synoptic diagram of Jurkat cell strain before and after the CD40L up-regulated, as shown in Figure 3: the average fluorescent strength that the Jurkat cell is combined with PEG fluorescent lipid body is 9.6, the average fluorescent strength of being combined with contrast polypeptide fluorescent lipid body is 8.8, and the average fluorescent strength of being combined with JD6 polypeptide fluorescent lipid body is 13.4.JD6 polypeptide fluorescent lipid body is the strongest with the combination of normal Jurkat cell, and also more weaker than PEG fluorescent lipid body with the combination of the contrast polypeptide fluorescent lipid body of same electric charge, be because itself with negative charge hindered with the surface equally with the cohesive process of the cell of negative charge.Verified that again even the situation that exists electric charge to disturb, JD6 polypeptide fluorescent lipid body still has stronger combination with the Jurkat cell.Explanation is on cell levels, and the fluorescent lipid body of JD6 has stronger binding ability with the cell of expressing CD40L.Raise the CD40L molecule after the expression of cell surface, the combination of PEG fluorescent lipid body and contrast polypeptide fluorescent lipid body and cell does not increase, and slightly descends on the contrary; But the Jurkat Cell binding intensity of CD40L up-regulated has increased greatly after JD6 fluorescent lipid body and this stimulation, obviously prompting JD6 fluorescent lipid body is proportionate with the bonding strength of cell and the expression of CD40L, strong explanation the combination of the liposome of JD6 modification and Jurkat cell be that the interaction of dependence JD6 and CD40L is carried out.To sum up experiment illustrate that the liposome of polypeptide JD6 and modification thereof and the cell of expressing CD40L have binding specificity, and this binding specificity is relevant with CD40L.
(2) separation of person joint's hydrops medium size lymphocyte and culture experiment
1) gets rheumatoid arthritis people's hydrarthrosis, add the Unidasa of 100U/ml, 37 degree hydrolysis 15 minutes; 2) hydrarthrosis 1 volume after will being hydrolyzed and the PBS liquid mixing of 1 volume add centrifuge tube, the human lymphocyte parting liquid of covering 1 volume it on, centrifugal 15 minutes of room temperature 1500rmp, white cell endless belt between the collecting layer; 3) lymphocyte of 1640 substratum 300g washing collection in centrifugal 10 minutes; 4) containing 10%FBS, penicillin (100units/ml), Streptomycin sulphate (100mg/ml), the RPMI1640 substratum in cultivate, 37 the degree, 5%CO
2
Fig. 4 contains the fluorescence intensity synoptic diagram that the fluorescent lipid body of 2% polypeptide lipid is combined with rheumatoid arthritis person joint hydrops medium size lymphocyte, as shown in Figure 4: in the combination experiment for 10 routine patient's hydrarthrosis medium size lymphocytes, the non-specific binding that has equally PEG liposome and cell, but it is stronger with the lymphocytic combination of these high expression levels CD40L to contain the liposome that JD6 modifies, and than the contrast polypeptide height of upsetting sequence.This figure has reflected the mean value that 10 routine patient's hydrarthrosis medium size lymphocytes are combined with the fluorescent lipid body.Although the cellular expression levels between the different patients is different, but JD6 fluorescent lipid body has still shown the higher bonding strength of contrast group, this illustrates that again the lymphocyte that raises liposome that JD6 modifies and the CD40L that separates has binding specificity from human body, and this binding ability is the interaction mediation by polypeptide JD6 and CD40L.
(3) separation of the spleen lymphocyte of mouse and culture experiment
1) mouse is taken off neck and put to death, be soaked in 75% the ethanol 5 minutes; 2) in super clean bench, take out mouse spleen, cross 200 eye mesh screens, add 4-5mL EZ-Sep
TMMouse 1 * lymphocyte separation medium grinds; 3) will hang the parting liquid that spleen cell is arranged and transfer to immediately in the 15mL centrifuge tube, cover 200 μ l RPMI1640 substratum on it; 4) room temperature 800g is centrifugal 30 minutes, and acceleration and the deceleration of whizzer is made as 3, it is slowly accelerated and slows down, and improves separating effect; 5) after the centrifugal end, can see at 1640 substratum and lymphocyte separation medium interface the white endless belt of clear densification, be buffy coat.With this confluent monolayer cells of aseptic straw sucking-off, add again the 10ml1640 substratum, the mixing washing; 6) room temperature 300g is centrifugal 10 minutes, abandons supernatant, cell is resuspended in 1640 substratum cell counting; 7) containing 10%FBS, penicillin (100units/ml), Streptomycin sulphate (100mg/ml), the RPMI1640 substratum in cultivate, 37 the degree, 5%CO
28) use TPA and Ionomycine to stimulate, raise the expression of its CD40L.Use the TPA of 6ng/ml and the Ionomycine of 1 μ g/ml to stimulate spleen lymphocyte, 37 degree 5%CO in containing the RPIM1640 substratum of 20%FBS
2Continue to cultivate 6 hours, detect the expression level of its CD40L and carry out subsequent experimental.
Fig. 5 is fluorescent lipid body and the fluorescence intensity synoptic diagram of mouse spleen lymphocyte before and after the CD40L up-regulated that contains 2% polypeptide lipid, as shown in Figure 5: because the CD40L in mouse source and the CD40L in people source have higher similarity, so can investigate JD6 fluorescent lipid body in conjunction be combined situation with the CD40L in mouse source with the lymphocyte of mouse spleen.When mouse lymphocyte was in quiescent condition, the cell of the CD40L positive was less, and the expression amount on the cell is lower.Use various fluorescent lipid bodies and tranquillization lymphocytic similar in conjunction with situation, the PEG liposome has certain non-specific binding, JD6 with and the contrast polypeptide liposome of upsetting sequence also not significance in conjunction with improving.After using TPA and Inomycine to stimulate lymphocyte, the up-regulated of CD40L, PEG fluorescent lipid body and contrast polypeptide fluorescent lipid body and these lymphocytic bonding strengths do not change, and the combination of JD6 fluorescent lipid body has increased significantly, illustrate that the JD6 modification can promote liposome to the combination of high expression level CD40L cell, can judge that thus the liposome of polypeptide JD6 and modification thereof also has specific binding based on CD40L for the cell of expressing mouse source CD40L.
(4) the CD40L target polypeptide is modified fluorescent lipid body and the investigation of Cell binding ability
1) get various cells to be measured, 1640 substratum 300g of 4 degree precoolings washed one time in centrifugal 10 minutes, and adjusted cell concn to 10 with 1640 substratum of precooling
6Individual/ml; 2) add all kinds of fluorescent lipid bodies (common PE G liposome, JD6 modified liposome, JD6c modified liposome) that contain identical PEG per-cent of 10 μ M in the various cells to be measured, 4 degree lucifuges were hatched 2 hours; 3) centrifugal 10 minutes washed cells of the PBS300g of precooling on ice wash 3 times, use the PBS re-suspended cell; 4) FACS detects FL-1 passage fluorescence intensity, judges fluorescent lipid body and Cell binding degree.
Fig. 6 contains the fluorescence intensity synoptic diagram that the fluorescent lipid body of different ratios polypeptide lipid is combined with the EAE mouse spleen lymphocyte, as shown in Figure 6: because the lymphocyte in the EAE in mice model body is high expression level CD40L, so extract its lymphocyte, whether the liposome that can investigate the JD6 modification in animal disease model has binding specificity for CD40L.Can see among the figure that the liposome that 0.5%JD6 modifies shows the high bond strength that is different from PEG and contrast polypeptide liposome.PEG fluorescent lipid body under the same percentage composition and the combination rate of EAE mouse lymphocyte are that the fluorescent lipid body that 6%, JD6 modifies is 14%, and contrast polypeptide fluorescent lipid body is 8%.Increase the content to 2% of JD6 in liposome, can see that liposome and lymphocytic combination have been increased to 33% significantly, and corresponding PEG fluorescent lipid body and lymphocytic combination rate do not have considerable change.Contrast simultaneously polypeptide fluorescent lipid body and lymphocytic combination rate be lower than its when 0.5% content in conjunction with level, analysis should be because polypeptide itself is electronegative, increase the content of polypeptide lipid in liposome so that liposome gets the electronegativity increase, cause having weakened with same electronegative Cell binding.But the fluorescent lipid body of modifying with the JD6 of same negative charge amount does not reduce because of the increase of negative charge with the lymphocytic combination of EAE of high expression level CD40L, has strengthened greatly on the contrary.This shows, in the cell experiment of mouse disease model, still observe the liposome of JD6 and modification thereof specific by CD40L in conjunction with the lymphocytic situation of CD40L high expression level.
The molecular targeted peptide modified methotrexate liposome of embodiment 4, CD40L
Present embodiment relates to a kind of methotrexate liposome, and this methotrexate liposome is the molecular targeted peptide modified methotrexate liposome of CD40L, and its preparation method comprises the steps:
1) according to molar ratio DPPC(DPPC): CHOL:JD6-PEG-DSPE(DSPE-PEG2000) (JD6-distearyl glyceryl phosphatide thanomin Macrogol 2000)=56:42:2,55:20:0.1,70:30:3.5 or 80:45:5 take by weighing each material;
2) DPPC and CHOL fully are dissolved in the trimethyl carbinol, dropwise add afterwards the JD6-PEG-DSPE aqueous solution in the lipid t-butanol solution, the keeping system clarification; In the preparation process of the contrast liposome that contains DSPEPEG2000, directly DSPE-PEG2000 and other lipids are dissolved in the trimethyl carbinol;
3) mixing polypeptide lipid and other compositions, quick freezing in liquid nitrogen places Freeze Drying Equipment-70 degree centigrade freeze-drying;
4) methotrexate is dissolved among the PBS, and concentration is 1mg/ml; Use is heated to 60 degrees centigrade the rapid hydrated lipidic component of methotrexate solution, and liposome is made in ultrasonic 30 seconds of 60 degrees centigrade of water-baths; The methotrexate that adds and the mol ratio of JD6-PEG-DSPE are respectively 10:2,1:0.1,200:3.5 or 400:5.
5) liquid nitrogen is 1 minute, and 3 minutes multigelation liposomes of 60 degrees centigrade of water-baths through 5 cycles, are made into large unilamellar liposome, improves liposome encapsulation;
6) use and to be heated to 60 degrees centigrade and must to extrude instrument liposome is crossed the 200nm polycarbonate membrane 11 times;
7) use the laser particle analyzer dynamic light scattering method to detect liposome particle diameter and distribution;
8) it is degassed CL-4B sepharose PBS to be washed twice rear rotary evaporation, filling 20ml chromatographic column, and PBS is as 10 column volumes of flushing in moving phase 8.4ml/ minute;
9) add 200 μ l and do not contain the saturated gel column of methotrexate blank liposome, liposome goes out the peak in the time of 5 minutes, continues 10 column volumes of flushing;
10) 200 μ l liposome application of sample to be purified keeps flow velocity, and the liposome peak when collecting 5 minutes is the MTX liposome behind the purifying;
11) continue wash-out, until free methotrexate goes out the peak, continue 10 column volumes of flushing, continue other liposomes of loading purifying.
The sign of the molecular targeted peptide modified methotrexate liposome of CD40L is as shown in table 1 below behind the purifying:
Table 1
| Median size (nm) | Polydispersity coefficient | The methotrexate encapsulation rate | |
| Common methotrexate liposome | 174.4 | 0.025 | 22.38% |
| JD6 modifies the methotrexate liposome | 193.9 | 0.117 | 20.16% |
As shown in Table 1: the methotrexate liposome of preparation extrude through the 200nm polycarbonate membrane and the sepharose purifying after, the particle diameter of common methotrexate liposome is about 180nm, polydispersity coefficient is very little.The non-common methotrexate liposome of particle diameter ratio of the methotrexate liposome that polypeptide JD6 modifies is bigger, is about 190nm, the more common methotrexate liposome that distributes slightly wide (polydispersity coefficient is 0.117).This is that polypeptide fragment is so that the hydration layer of liposome is large than the liposome that does not contain polypeptide, so the median size that the use laser scattering method measures will be more greatly because the surface of the methotrexate liposome that polypeptide JD6 modifies has peptide modifiedly.Illustrate that the methotrexate liposome that polypeptide JD6 modifies is successfully prepared, its outside surface is modified with the JD6 polypeptide fragment really.Can see simultaneously, the methotrexate liposome that JD6 modifies to the encapsulation rate of medicine methotrexate than non-peptide modified low a bit, but there is no very large difference, can think that methotrexate liposome and common methotrexate liposome that JD6 modifies there is no there was no significant difference aspect the medicine parcel, both can be contrasted to use and carry out follow-up research.
Compliance test result:
(1) cell culture experiments
Cell culture condition is as follows: Jurkat, containing 10%FBS, penicillin (100units/ml), Streptomycin sulphate (100mg/ml), Jurkat cell 1640 substratum in cultivate, 37 the degree, 5%CO
2Carry out afterwards the cytotoxicity of the molecular targeted peptide modified methotrexate liposome of CD40L and investigate, comprise the steps:
1) according to every hole 1*10
4Individual cell is inoculated in 96 orifice plates, the substratum of the corresponding cell of every hole 100 μ l;
2) in each hole, add respectively different concns the methotrexate liposome modified of JD6 and the common methotrexate liposome with identical PEG percentage composition, 3 multiple holes of each drug level;
3) each hole supplemented medium, the volume of adjusting in every hole is consistent, follow-up 37 degree 5%CO2 culturing cells 68 hours;
4) add 10 μ l CCK reaction substrates in each hole, 37 degree 5%CO2 culturing cells 3 hours;
5) microplate reader detects each hole OD450 value, and setting 570nm is reference wavelength;
6) as 100%, the quantity of cell in each hole of converting is calculated cell survival per-cent, investigates the cytotoxicity of respectively organizing medicine without the control cells OD450 of medicine group in use.
As shown in Figure 7, as shown in Figure 7: the methotrexate liposome that common methotrexate liposome and JD6 modify kills and wounds the degree of expressing the CD40L cell and becomes positive correlation with the amount of its adding, and along with the increase of medicine Concentration of Methotrexate, the survival rate of cell reduces gradually.In drug level lower (6nM is to 30nM), the cytotoxicity of the methotrexate lipid that polypeptide JD6 modifies is greater than common methotrexate liposome, this explanation polypeptide methotrexate liposome is owing to exist the JD6 targeting peptides, so that for the Jurkat cell in conjunction with grow, when drug level is low, according to the interaction of JD6 on the liposome and cell surface CD40L, can kill and wound more targetedly the target cell of expressing CD40L.When drug level increases, the free drug that peptide modified methotrexate liposome and common methotrexate liposome discharge in culturing process all is enough to kill and wound a large amount of cells (cell survival rate is down to about 30%), thereby in drug level higher (60nM is to 120nM), the cytotoxicity of two kinds of liposomes does not have significant difference.But under lower concentration, kill and wound specificity, illustrate in treatment and just might use medicine still less to reach same result for the treatment of by the liposome drug-loading system of JD6 target mediation preparation.
(2) the molecular targeted peptide modified methotrexate liposome of CD40L is to the treatment checking of EAE mouse
1, the foundation of EAE mouse model comprises the steps:
1) polypeptide myelin oligodendrocyte glycoprotein 35-55 is dissolved among the PBS, and concentration is 2mg/ml; PT is dissolved among the PBS, and concentration is 2 μ g/ml;
2) remove isopyknic MOG35-55 solution and CFA mixing, be prepared into emulsion in Three-way injector, each is injected at 100 μ l emulsions near the inguinal lymph nodes of the C57/BL6 mouse left and right sides, and timing is the 0th day of modeling;
3) injection is rear the 1st day, mouse after the immunity is carried out the PT tail vein injection, every 100 μ l;
4) observe the mouse invasion situation every day, mark for mouse according to 5 point-score standards of grading (Kono method).Classification is as follows: 0 is divided into and does not fall ill; 1 to be divided into tail unable; 2 are divided into slight rear myasthenia of limbs; 2.5 being divided into one-sided hind leg seriously benumbs; 3 are divided into the bilateral hind leg seriously benumbs; 3.5 be divided into the serious paralysis of bilateral hind leg and one-sided front acroparalysia; 4 are divided into tetraplegia; 5 be divided into dying or dead;
5) draw the morbidity score curve, calculate the morbidity ratio, investigate the modeling situation, as shown in Figure 8, as shown in Figure 8: the symptom of typical EAE modeling success occurs when mouse after, timing was carried out drug treatment for the 0th day after treating to it, investigated the targeted therapy effect of the peptide modified methotrexate liposome of JD6.Each organizes the initial average mark of EAE mouse all is 2 minutes, and PBS group can be indicated in the situation without pharmacological agent as the blank group, can see the prolongation along with the time, and the incidence of mouse is serious gradually, and the disease score curve is the trend that rises gradually.Can see that in using common methotrexate liposome therapeutic group the morbidity scoring of EAE mouse all is lower than the PBS control group, illustrate still has significant curative effect after use methotrexate liposome is treated mouse.The methotrexate liposome that JD6 is peptide modified and the curative effect of common methotrexate liposome relatively can see that the result for the treatment of of the methotrexate liposome that JD6 is peptide modified all is better than common methotrexate liposome.Illustrate because it has the effect of CD40L target, can specific target and suppress to cause in the EAE mouse CD40L high expression level T cell of excessive inflammation.Consider that the PEG on the common methotrexate liposome also has some nonspecific lymphsystem Targeting Effects, the result for the treatment of of the methotrexate liposome that JD6 is peptide modified still can significantly be better than common methotrexate liposome, that is to say in the body of animal disease model in the experiment, the effect of the drug-loaded liposome target high expression level CD40L lymph T cell of JD6 mediation is strong, and the disease that causes for the CD40L high expression level has better therapeutic action.
2, the molecular targeted peptide modified methotrexate liposome of CD40L comprises the steps: the result for the treatment of verification method of EAE mouse
1) according to EAE mouse invasion curve, when modeling in the time of the 18th day, the mouse invasion scoring tends towards stability, and prompts for modeling success administration time point;
2) according to the methotrexate dosage tail vein injection administration of 2mg/Kg, administration in the 18th day after modeling.Mouse is divided into three groups, every group of 10 mouse, each organizes average mark all is 2 minutes, and one group of methotrexate liposome of modifying to JD6 is given the common methotrexate liposome with identical PEG percentage composition for one group, and one group to physiological saline, was designated as administration the 1st day;
3) follow-up respectively administration in the 4th, 8,12,15,18 day;
4) give the scoring of EAE mouse in per 3 days, observe the development and change of its state of an illness;
3, EAE Mice Body endolymph cell CD40L expression level changes investigation
Sacrificed four mouse (every scoring is 2 minutes) respectively at the 10th day for every group, the PBMC in its blood of separation and purification, to its CD3, the CD40L molecule carries out mark, and FACS detects various cellular component changing conditions.As shown in Figure 9, as shown in Figure 9: the percentage composition of CD40L positive T cell can directly reflect the T the cell whether peptide modified methotrexate liposome of JD6 kills and wounds high expression level target protein CD40L targetedly in the peripheral blood lymphocyte.Can see that normal mouse is about 0.01% in peripheral blood CD40L positive cell rate, and CD40L positive cell ratio is higher in EAE model mice (the disease average mark 2 minutes) peripheral blood, is 0.4%, exceeds very many than normal value.After using the methotrexate liposome mouse to be carried out 3 treatments, to treating mid-term, can see that significantly changing appears in the level of CD40L positive cell in the Mouse Peripheral Blood Lymphocyte.Methotrexate liposome and common methotrexate liposome that JD6 modifies have all reduced the CD40L positive rate.And the methotrexate liposome that JD6 modifies is all good than common methotrexate liposome to the reduction effect of CD40L positive rate.That is to say that by JD6 target mediation the methotrexate liposome after peptide modified is better than non-targeted liposome to the fragmentation effect of CD40L high expression level T cell, this advantage is embodying clearly just in mid-term for the treatment of.The drug-loaded liposome of the CD40L target of the method preparation can at the CD40L high expressing cell that suppresses to cause the disease reason, be reached the effect of better healing disease.
4, the separation and detection of the spleen of mouse and lymphoglandula medium size lymphocyte
1) eye socket is got blood;
2) peripheral blood is placed 15mL centrifuge tube lower floor, the upper strata adds the parting liquid of equal-volume lymphoglandula (health both sides inguinal lymph nodes and health both sides axillary lymph knot) cell, centrifugal 30 minutes of room temperature 800g, acceleration and the deceleration of whizzer are made as 3, it is slowly accelerated and deceleration, improve separating effect;
3) after the centrifugal end, can see at 1640 substratum and lymphocyte separation medium interface the white endless belt of clear densification, be buffy coat.With this confluent monolayer cells of aseptic straw sucking-off, add again the 10ml1640 substratum, the mixing washing;
4) room temperature 300g is centrifugal 10 minutes, abandons supernatant, cell is resuspended in 1640 substratum cell counting;
5) collect various cells to be measured, the FACS damping fluid re-suspended cell of precooling on ice uses the centrifuging washed twice, adjusts cell concn to 10
6Individual/100 μ l;
6) add fluorescently-labeled CD3 in the cell in mouse source, CD40L antibody, mixing, 4 ℃ of lucifuges were hatched 30 minutes, the FACS washed twice of precooling, 1% Paraformaldehyde 96 stationary liquid is resuspended, adjusts concentration to 10
6Individual/1ml;
7) FACS detects the expression degree of corresponding fluorescently-labeled each molecule in the FL-1/FL-2 passage.
In sum, of the present invention have with polypeptide that the cell-specific of expressing the CD40L molecule is combined with after the DSPEPEG material is connected, be assembled on the liposome, the liposome of this target polypeptide modification external can well with the Cell binding of expression CD40L.Modify the liposome of parcel methotrexate with polypeptide DSPE-PEG connecting material, can in the activity of external more effective inhibition CD40L positive cell, in the experimental autoimmune encephalomyelitis animal model, show result for the treatment of simultaneously.
Claims (8)
1. a ligand polypeptide of being combined with the CD40L protein-specific is characterized in that, comprises the aminoacid sequence shown in the SEQ ID NO.1.
2. ligand polypeptide of being combined with the CD40L protein-specific according to claim 1 is characterized in that, described ligand polypeptide contains more than or equal to two aminoacid sequences shown in SEQ ID NO.1.
3. ligand polypeptide of being combined with the CD40L protein-specific according to claim 1 is characterized in that, described ligand polypeptide comprises sequence shown in the SEQ ID NO.1, and adds linking group or connection aminoacid sequence at its C-terminal and/or N-terminal.
4. sequestered polypeptide, pattern of fusion polypeptide, chimeric class polypeptide or polymkeric substance take ligand polypeptide as monomer that ligand polypeptide of being combined with the CD40L protein-specific as claimed in claim 1 forms.
5. a delivery system is characterized in that, this system comprises: ligand polypeptide of being combined with the CD40L protein-specific as claimed in claim 1, drug-loading system, at least a active substance.
6. delivery system according to claim 5, it is characterized in that described ligand polypeptide is connected to following arbitrary body structure surface: nanoparticle, polymkeric substance, liposome, vesica, solid lipid particulate, micella, carbon nanotube, organoid, lipoprotein.
7. delivery system according to claim 5, it is characterized in that, described drug-loading system is liposome, its moiety comprises: phosphatidylcholine, phosphatidylethanolamine, phosphatidyl glycerol, phosphatidylinositols, phosphatidylserine, phosphatidic acid, Val, sphingophospholipid, cerebroside, Sphingolipids,sialo, ergosterol, cholesterol, cation lipid, lysophospholipid, polyethyleneglycol modified lipid, the lipid that ligand polypeptide is modified.
8. delivery system according to claim 5, it is characterized in that, described at least a active substance is: antitumor drug, antimetabolite, the spindle body toxic alkaloid, cytotoxicity/antitumor antibiotics, topoisomerase enzyme inhibitor, photosensitizers, kinase inhibitor, microbiotic, antibiotic, anti-inflammatory drug, immunosuppressor, anti-infectives, antiviral, insect repellent, antiparasitic compound, be used for ultrasonic contrast, pneumoradiography, the diagnostic reagent of nuclear medicine contrast medium, goal gene, the antisense oncogene, suicide gene, apoptosis gene, cytokine gene, or the combination of said gene, or contain the carrier for expression of eukaryon DNA of said gene.
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| CN101855237A (en) * | 2007-11-05 | 2010-10-06 | 上海交通大学 | Peptide ligand directed drug delivery |
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