CN102875647B - 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 protein and peptide technical field, relate in particular to a kind of ligand polypeptide and delivery system of being combined with CD40L protein-specific.
Background technology
CD40L is tumour necrosis factor (TNF) superfamily member, is II type membranin, mainly expresses at CD4+T cell surface, comprises Th0, Th1 and Th2 cell subsets.CD40L also expresses at other lymphocytic cell surfaces, as CD8+T cell or the B cell surface of activation.Research shows, functional CD40L also expresses non-lymphocyte in hematopoiesis system, as the surface of the platelet cell of activation.The T cell of activation can, at transient expression CD40L in latter tens minutes of activation, reach peak at 6 hours, expression amount starts to decline subsequently.
CD40/CD40L is a pair of important costimulatory molecules in 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 B cell because Fas(CD95) or the crosslinked apoptosis causing of IgM of antigen induction; The feminine gender screening of induction B cell etc.
CD40/CD40L signal path is relevant to a lot of autoimmune disorders, and it can cause body to produce immunopathogenesis reaction extremely.Lymphocyte CD 40L expression amount calibration ordinary person level in rheumatoid arthritis (RA) patient synovial fluid increases.In multiple sclerosis (MS), the situation of CD40L high expression level detected equally.Compared to 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.Due to CD40/CD40L signal, to pass to path relevant to disease of immune system, and large quantity research completes the treatment to disease for this signal path of blocking-up.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 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, lower the expression of ICAM adhesion molecule, reduce monocyte and produce NO etc.In follow-up relevant experimentation on animals and clinical experiment, find that mechanism that anti-CD40L antibodies plays a role is except blocking-up CD40/CD40L signal path, also comprise that antibody Fc section mediation removes the CD40L+T cell of activation outward.
Enter several antibody of clinical experiment, all because the situation that has occurred thrombus has been suspended clinical trial.The formation of this thrombus may be because of the FcRIIA receptors bind on thrombocyte in the Fc section on anti-CD40L antibodies and blood, causes being cross-linked between thrombocyte, causes blood aggegation to occur embolism.The improvement that follow-up antagonist on this basis carries out, removal can cause the Fc section of thrombus, only retains the Fab section on 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 cannot reach the effect of CD40L+T cell clearance.
For defect and the limitation of the antibody drug of blocking-up CD40/CD40L signal path, can further transform its Research Thinking: 1. screening is for the ligand polypeptide of CD40L, Fab section using this polypeptide as antibody is used, and reaches the effect of blocking-up CD40/CD40L signal path.2. in conjunction with target ligand polypeptide and the liposome drug delivery technologies of CD40L, the cell by polypeptide target to high expression level CD40L, releases cell toxicity medicament targetedly, reaches the effect that suppresses CD40L+T cell proliferation.The existing trimerization polypeptide for CD40 occurs at present, and on cell levels, effectively suppress CD40/CD40L signal path, but there is not experiment report in further body, do not have in addition for the ligand polypeptide of CD40L yet and realize the report that blocking-up CD40/CD40L signal is gone the same way.
Liposome be with the double-deck phospholipid molecule similar of microbial film 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, through the modification of the contour hydrophilic pair of property molecule of DSPE-PEG2000, extends its cycling time in blood, increases drug release also action time.By targeted molecular, as polypeptide, monoclonal antibody etc. are connected on liposome simultaneously, make and there is 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 liposome internalization, to promote that medicine discharges in cell, thereby improve drug effect.
Thus, CD40L target polypeptide is combined with 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 CD40L protein-specific.This ligand polypeptide is connected with distearyl glyceryl phosphatide thanomin polyoxyethylene glycol maleimide, make target synthetic materials, then use target synthetic materials further to prepare target liposomes, measure target synthetic materials and the impact of target liposomes on inside and outside cell and CD40L high expression level disease model.
The object 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 CD40L protein-specific, comprises the aminoacid sequence shown in SEQ IDNO.1.
Preferably, described ligand polypeptide contains and is more than or equal to two aminoacid sequences as shown in SEQ ID NO.1.
Preferably, described ligand polypeptide comprises sequence shown in 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 taking ligand polypeptide as monomer that a kind of aforesaid ligand polypeptide of being combined with 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 CD40L protein-specific, drug-loading system, at least one 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 one 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, for ultrasonic contrast, pneumoradiography, the diagnostic reagent of nuclear medicine contrast medium, goal gene, antisense oncogene, suicide gene, apoptosis gene, cytokine gene, or the combination of said gene, or the carrier for expression of eukaryon DNA that contains said gene.
The present invention has following beneficial effect:
(1) after of the present invention and ligand polypeptide modified tetramino that CD40L protein-specific is combined acid connecting joint, be connected to synthetic materials with distearyl glyceryl phosphatide thanomin polyoxyethylene glycol maleimide, the fluorescent lipid body being prepared into shows and is well combined effect with the cell of CD40L high expression level in vitro; Through fluorescent substance mark, the distribution in vivo of this polypeptide is basic to coincide with the lymphoglandula that is rich in T cell.
(2) after this ligand polypeptide modified tetramino acid connecting joint, be connected to synthetic materials with distearyl glyceryl phosphatide thanomin polyoxyethylene glycol maleimide, the methotrexate liposome being prepared into shows stronger lethal effect to the cell of the CD40L positive in vitro, has shown better result for the treatment of in 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 CD40L protein-specific of the present invention not only has advantages of traditional antibody molecule, and molecular weight is little, tissue penetration is strong, there is good CD40L targeting, can be developed as early diagnosis kit and specific medicine for the autoimmune disorder of some CD40L high expression levels, this polypeptide is combined and can also be served as target medicine carrier with liposome simultaneously, develop into the targeted drug delivery system for autoimmune disorder, realize stronger result for the treatment of.
Brief description of the 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 be containing the fluorescent lipid body of 2% polypeptide lipid in conjunction with Jurkat cell strain the fluorescence intensity schematic diagram before and after CD40L up-regulated;
Fig. 4 is the fluorescence intensity schematic diagram of being combined with rheumatoid arthritis person joint hydrops medium size lymphocyte containing the fluorescent lipid body of 2% polypeptide lipid;
Fig. 5 is the fluorescence intensity schematic diagram before and after CD40L up-regulated containing the fluorescent lipid body of 2% polypeptide lipid and mouse spleen lymphocyte;
Fig. 6 is the fluorescence intensity schematic diagram of being combined with EAE mouse spleen lymphocyte containing the fluorescent lipid body of different ratios polypeptide lipid;
Fig. 7 is that the cytotoxicity to Jurkat cell strain of the molecular targeted peptide modified methotrexate liposome of CD40L is investigated situation schematic diagram;
Fig. 8 is that EAE mouse is used the molecular targeted peptide modified methotrexate liposome therapeutic clinical score changing conditions schematic diagram of CD40L;
Fig. 9 is that EAE mouse is used after CD40L molecular targeted peptide modified methotrexate liposome therapeutic CD40L positive T cell composition changing conditions schematic diagram in peripheral blood.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.These embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, for example Sambrook equimolecular clone: 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 CD40L protein-specific, applicant has investigated the binding ability of this peptide modified liposome and CD40L high expressing cell simultaneously and has carried out external qualification, prepare fluorescently-labeled polypeptide it is carried out to the investigation distributing in body, confirm the ability of this polypeptide and CD40L high expressing cell specific binding.The process that the CD40L molecular specificity target polypeptide (ligand polypeptide of being combined with CD40L protein-specific) the present invention relates to obtains is as shown in Figure 1:
One, computer-aided screening CD40L target ligand candidate polypeptide
Use the dawn A4000 of Shanghai computer center to calculate unit computing, build protein binding model based on CD40/CD40LX ray diffraction crystalline structure, build and the incorporative peptide library of CD40L, the polypeptide ligand molecule of screening target CD40L.
1) use SPDBV3.7, manually produce CD40L dimeric structure, for combination screening
2) PSCAN2.2.2 scanning dimeric structure surface, produces the pocket that is applicable to combination
3) polypeptide fragment of the upper corresponding binding pocket of CD40 position is carried out to preliminary screening, use Autodock3.0.5 to utilize Lamarckian genetic algorithm calculations incorporated energy with docking energy and by its sequence
4) analyze the hydrogen bond contribution of the each amino acid of candidate's polypeptide to combination, candidate's polypeptide is carried out to point mutation, generate the skewed popularity peptide library that has stronger binding ability with CD40L dimer, still use Autodock3.0.5 to utilize Lamarckian genetic algorithm calculate in storehouse all polypeptide in conjunction with energy and docking energy and sort, acquisition CD40L target polypeptide candidate peptide.
Two, the SPR of CD40L candidate polypeptide ligand screening
Use the BIAcore X100 of GE healthcare, CM5 chip, uses competition combined techniques, and the signal attenuation degree producing by surface plasma resonance technology is screened the polypeptide ligand molecule of target CD40L in albumen aspect.
1), according to chip operation instruction, hCD40 is fixed on CM5 chip
2) in moving phase, add hCD40L, investigate the combination degree of signal response with reflection CD40L and CD40
3) in moving phase, add hCD40L and a certain proportion of candidate's polypeptide simultaneously, investigate signal response
4) according in conjunction with response signal intensity, calculate each candidate's peptide to the competition per-cent of CD40L sequence, obtain CD40L target polypeptide.
Three, peptide sequence is synthetic
Candidate's polypeptide and CD40L targeted polypeptide ESEEED(are called for short JD6, as shown in SEQ ID NO.1), the control peptide DSDDDE(with identical charges number is called for short JD6c) and the ESEEEDGGGC(that contains halfcystine end of the design for the convenient chemistry of subsequent experimental connects as shown in SEQ ID NO.2) and DSDDDEGGGC polypeptide synthetic by Chengdu Kai Jie biological medicine Development Co., Ltd, all pass through HPLC purifying and Mass Spectrometric Identification, purity is greater than 95%, and molecular weight conforms to theoretical value.
derivative polypeptide of embodiment 1, CD40L targeted polypeptide and preparation method thereof
The present embodiment relates to the derivative polypeptide 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), 0.8mg polypeptide is dissolved in 400 μ l DMSO;
2) equal-volume adds 15 μ l triethylamines after mixing Cy5.5-NHS and polypeptide in system, is placed in isothermal vibration reactor, room temperature, and lucifuge reaction is spent the night;
3) to be placed in molecular weight cut-off be 1000 dialysis tubing to reaction system, uses lucifuge dialysis under PBS room temperature, changes water three times, removes DMSO, triethylamine and free Cy5.5-NHS;
4) the CY5.5-polypeptide after purifying keeps in Dark Place in-80 degrees Celsius.
Compliance test result, detects polypeptide JD6 and contrast polypeptide JD6c distribution situation in vivo thereof.Effect process is as follows: polypeptide-Cy5.5 solution of aforementioned preparation is entered in Mice Body by tail vein injection, the amount of every injected in mice Cy5.5 mark is 10ug, after injection according to different time points, utilize the distribution situation of small animal living body fluorescence imaging system scan polypeptide in Mice Body, scanning result utilizes the analysis software that system carries to process and analyze, as shown in Figure 2, as shown in Figure 2: polypeptide is through after Cy5.5 fluorescent mark, and in the body of fluorescence, distribution represents polypeptide distribution situation in vivo.In figure, use the pseudo-color fluorescence intensity that represents, 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 normalized, the convenient polypeptide of investigating each position of different time points mouse distributes and aggregation extent.In Fig. 2, can see polypeptide JD6 in vivo prolongation be in time enriched to gradually the lymphoglandula at upper limbs neck chest place, and hold more than 6 hours in these lymphoid organs are protected.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, illustrate that contrast polypeptide removed by health very soon, do not rest on lymphoglandula, it is that to have 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 lymphsystem.
embodiment 2, there is the synthetic materials of CD40L targeting
The 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 and is obtained by the derivative polypeptide of CD40L molecular specificity target polypeptide.The sequence of described CD40L molecular specificity target polypeptide is ESEEED, when synthetic, adds GGGC at C end, and then obtains derivative polypeptide, and its sequence is ESEEEDGGGC; On the halfcystine of this derivative polypeptide, free sulfydryl just reacts with maleimide base group and is connected to DSPE-2000-Maleimide(distearyl glyceryl phosphatide thanomin Macrogol 2000 maleimide, polyoxyethylene glycol wherein also can be selected PEG5000) above, obtain having the synthetic materials of 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 CD40L targeting.
The preparation of the synthetic materials (wherein polypeptide is CD40L molecular specificity target polypeptide) of the described CD40L of having targeting comprises the steps:
1, weigh DSPE-PEG2000-Maleimide lipid, add chloroform to 10mg/ml, in eggplant-shape bottle, dissolve, nitrogen dries up, and forms lipid membrane;
2,1.5 times (molar ratios) are dissolved in to HEPES(hydroxyethyl piperazine second thiosulfonic acid buffer salt solution in the polypeptide powder of lipid under nitrogen protection) in, add in the eggplant-shape bottle that has lipid membrane;
3,, in isothermal reactor, the lower 10 degrees Celsius of reactions of nitrogen protection are spent the night;
4, reaction system is transferred in the dialysis tubing of WMCO1000, uses deionized water dialysed overnight three times, obtains having the synthetic materials of CD40L targeting, i.e. polypeptide-PEG-DSPE.
the molecular targeted peptide modified fluorescent lipid body of embodiment 3, CD40L
The 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): CHOL(cholesterol): fluorescein DHPE(is fluorescein-labeled 1,2-palmityl phosphatidylethanolamine)=60:40:1,70:30:1 or 80:20:1 take, be fully dissolved in the trimethyl carbinol;
2) in lipid t-butanol solution, dropwise add polypeptide-PEG-DSPE aqueous solution (prepared by embodiment 2), keeping system is clarified the molar ratio of peptide lipid and fluorescein DHPE at the most and is respectively 0.1:1,0.5:1,1:1,2:1,3:1,4:1,5:1;
3) mix polypeptide lipid and other lipid composition, quick freezing in liquid nitrogen, is placed in Freeze Drying Equipment-70 degree Celsius freeze-drying;
4) use the rapid hydrated lipidic component of PBS, ultrasonic 30 seconds of lucifuge water-bath, prepares liposome;
5) lucifuge use is extruded instrument liposome is crossed to 200nm polycarbonate membrane 11 times;
6) use laser particle analyzer dynamic light scattering method to detect liposome particle diameter and distribution.
Compliance test result:
(1) cell culture experiments
Jurkat, containing 10%FBS(peptide bovine serum), penicillin (100units/ml), Streptomycin sulphate (100mg/ml), Jurkat cell 1640 substratum in cultivate, 37 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, containing 37 degrees Celsius of 5%CO in 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 be containing the fluorescent lipid body of 2% polypeptide lipid in conjunction with Jurkat cell strain the fluorescence intensity schematic diagram before and after CD40L up-regulated, as shown in Figure 3: the average fluorescent strength that 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 the combination of contrast polypeptide fluorescent lipid body with same electric charge is also more weaker than PEG fluorescent lipid body, be because itself with negative charge hindered the cohesive process of cell with negative charge same with surface.Again verified, even the situation that exists electric charge to disturb, JD6 polypeptide fluorescent lipid body still has stronger combination with Jurkat cell.Illustrate on cell levels, the fluorescent lipid body of JD6 has stronger binding ability with the cell of expressing CD40L.Raise 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 declines on the contrary; But after JD6 fluorescent lipid body and this stimulation, the Jurkat Cell binding intensity of CD40L up-regulated has increased greatly, obviously prompting JD6 fluorescent lipid body is proportionate with the bonding strength of cell and the expression of CD40L, strong explanation the liposome of JD6 modification and the combination of 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 to CD40L.
(2) separation of person joint's hydrops medium size lymphocyte and culture experiment
1) get rheumatoid arthritis people's hydrarthrosis, add the Unidasa of 100U/ml, 37 degree hydrolysis 15 minutes; 2) hydrarthrosis 1 volume after hydrolysis and the PBS liquid of 1 volume are mixed, add centrifuge tube, on it, cover the human lymphocyte parting liquid of 1 volume, centrifugal 15 minutes of room temperature 1500rmp, white cell endless belt between collecting layer; 3) lymphocyte that 1640 substratum 300g washing in centrifugal 10 minutes is collected; 4) containing 10%FBS, penicillin (100units/ml), Streptomycin sulphate (100mg/ml), RPMI1640 substratum in cultivate, 37 degree, 5%CO
2.
Fig. 4 is the fluorescence intensity schematic diagram of being combined with rheumatoid arthritis person joint hydrops medium size lymphocyte containing the fluorescent lipid body of 2% polypeptide lipid, as shown in Figure 4: in the combination experiment for 10 routine patient's hydrarthrosis medium size lymphocytes, there is equally the non-specific binding of PEG liposome and cell, but the liposome that contains JD6 modification and the lymphocytic combination of these high expression levels CD40L are stronger, and higher than the contrast polypeptide of upsetting sequence.This figure has reflected the mean value that 10 routine patient's hydrarthrosis medium size lymphocytes are combined with fluorescent lipid body.Although the cellular expression levels between different patients is different, but JD6 fluorescent lipid body has still shown the bonding strength higher than control group, this liposome that again illustrates that JD6 modifies has binding specificity with the lymphocyte that the CD40L separating from human body raises, and this binding ability is to mediate by the interaction of polypeptide JD6 and CD40L.
(3) separation of the spleen lymphocyte of mouse and culture experiment
1) de-mouse neck is put to death, be soaked in 75% 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) there is the parting liquid of spleen cell to transfer to immediately in 15mL centrifuge tube by outstanding, on it, cover 200 μ l RPMI1640 substratum; 4) centrifugal 30 minutes of room temperature 800g, is made as 3 by the acceleration of whizzer and deceleration, makes it slowly accelerate and slow down, and improves separating effect; 5) after centrifugal end, can on 1640 substratum and lymphocyte separation medium interface, see the white endless belt of clear densification, be buffy coat.With this confluent monolayer cells of aseptic straw sucking-off, then add 10ml1640 substratum, mix washing; 6) centrifugal 10 minutes of room temperature 300g, abandons supernatant, cell is resuspended in 1640 substratum to cell counting; 7) containing 10%FBS, penicillin (100units/ml), Streptomycin sulphate (100mg/ml), RPMI1640 substratum in cultivate, 37 degree, 5%CO
2; 8) 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, containing 37 degree 5%CO in 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 the fluorescence intensity schematic diagram before and after CD40L up-regulated containing the fluorescent lipid body of 2% polypeptide lipid and mouse spleen lymphocyte, 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.At mouse lymphocyte, during in quiescent condition, the cell of the CD40L positive is less, and the expression amount on cell is lower.Use the lymphocytic combination situation of various fluorescent lipid bodies and tranquillization similar, PEG liposome has certain non-specific binding, JD6 with and the contrast polypeptide liposome of upsetting sequence also not the combination of significance improve.When using TPA and Inomycine to stimulate after 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 JD6 modification can promote the combination of liposome to high expression level CD40L cell, can judge thus, the liposome of polypeptide JD6 and modification thereof also has the specific binding based on CD40L for the cell of expressing mouse source CD40L.
(4) CD40L target polypeptide is modified fluorescent lipid body and the investigation of Cell binding ability
1) get various cells to be measured, centrifugal 10 minutes of the 1640 substratum 300g washing of 4 degree precoolings one time, and with 1640 substratum adjustment cell concns to 10 of precooling
6individual/ml; 2) in various cells to be measured, add all kinds of fluorescent lipid bodies that contain identical PEG per-cent of 10 μ M (common PE G liposome, JD6 modified liposome, JD6c modified liposome), 4 degree lucifuges are hatched 2 hours; 3) centrifugal 10 minutes washed cells of the PBS300g of precooling on ice, wash 3 times, use PBS re-suspended cell; 4) FACS detects FL-1 passage fluorescence intensity, judges fluorescent lipid body and Cell binding degree.
Fig. 6 is the fluorescence intensity schematic diagram of being combined with EAE mouse spleen lymphocyte containing the fluorescent lipid body of different ratios polypeptide lipid, as shown in Figure 6: because the lymphocyte in EAE in mice model body is high expression level CD40L, so extract its lymphocyte, whether the liposome that can investigate JD6 modification in animal disease model has binding specificity for CD40L.In figure, can see, 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 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 lower than it combination level when 0.5% content, analysis should be because polypeptide itself is electronegative, increase the content of polypeptide lipid in liposome and make liposome obtain electronegativity increase, cause having weakened with same electronegative Cell binding.But the fluorescent lipid body that the JD6 with same negative charge amount modifies and the lymphocytic combination of EAE of high expression level CD40L do not reduce because of the increase of negative charge, have strengthened greatly on the contrary.As can be seen here, 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
The 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 each material;
2) DPPC and CHOL are fully dissolved in the trimethyl carbinol, dropwise add afterwards the JD6-PEG-DSPE aqueous solution in lipid t-butanol solution, keeping system clarification; In the preparation process of the contrast liposome containing DSPEPEG2000, directly DSPE-PEG2000 and other lipids are dissolved in the trimethyl carbinol;
3) mix polypeptide lipid and other compositions, quick freezing in liquid nitrogen, is placed in Freeze Drying Equipment-70 degree Celsius freeze-drying;
4) methotrexate is dissolved in PBS, and concentration is 1mg/ml; Use is heated to the rapid hydrated lipidic component of methotrexate solution of 60 degrees Celsius, and ultrasonic 30 seconds of 60 degrees Celsius of water-baths, make liposome; The methotrexate adding and the mol ratio of JD6-PEG-DSPE are respectively 10:2,1:0.1,200:3.5 or 400:5.
5) liquid nitrogen 1 minute, 3 minutes multigelation liposomes of 60 degrees Celsius of water-baths, through 5 cycles, are made into large unilamellar liposome, improve liposome encapsulation;
6) use and be heated to 60 degrees Celsius and must extrude instrument liposome is crossed to 200nm polycarbonate membrane 11 times;
7) use laser particle analyzer dynamic light scattering method to detect liposome particle diameter and distribution;
8) CL-4B sepharose PBS is washed to twice rear rotary evaporation degassed, filling 20ml chromatographic column, PBS is as 10 column volumes of flushing in moving phase 8.4ml/ minute;
9) add 200 μ l not containing the saturated gel column of methotrexate blank liposome, 5 minutes time, liposome goes out peak, continues to rinse 10 column volumes;
10) 200 μ l liposome application of sample to be purified, keeps flow velocity, and the liposome peak while collecting 5 minutes, is the MTX liposome after purifying;
11) continue wash-out, until free methotrexate goes out peak, continue to rinse 10 column volumes, continue other liposomes of loading purifying.
After purifying, the sign of the molecular targeted peptide modified methotrexate liposome of CD40L is as shown in table 1 below:
Table 1
| Median size (nm) | Polydispersity coefficient | Methotrexate encapsulation rate | |
| Common methotrexate liposome | 174.4 | 0.025 | 22.38% |
| JD6 modifies methotrexate liposome | 193.9 | 0.117 | 20.16% |
As shown in Table 1: the methotrexate liposome of preparation extrude through 200nm polycarbonate membrane and 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 makes the hydration layer of liposome than the liposome containing polypeptide is not large, so the median size that uses laser scattering method to measure will be more greatly because the surface of 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 JD6 polypeptide fragment really.Can see simultaneously, the methotrexate liposome that JD6 modifies is peptide modified lower than non-to the encapsulation rate of medicine methotrexate, 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 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 degree, 5%CO
2.Carry 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 there is the common methotrexate liposome of identical PEG percentage composition, 3 multiple holes of each drug level;
3) each hole supplemented medium, adjusts volume in every hole consistent, follow-up 37 degree 5%CO2 culturing cell 68 hours;
4) in each hole, add 10 μ l CCK reaction substrates, 37 degree 5%CO2 culturing cells 3 hours;
5) microplate reader detects each hole OD450 value, and setting 570nm is reference wavelength;
6) use is without the control cells OD450 of medicine group as 100%, and the quantity of cell in each hole of converting, calculates cell survival per-cent, investigates the cytotoxicity of each group of medicine.
As shown in Figure 7, as shown in Figure 7: the methotrexate liposome that common methotrexate liposome and JD6 modify kills and wounds expresses the amount that the degree of CD40L cell adds with it and become positive correlation, 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 there being JD6 targeting peptides, make for the combination grow of Jurkat cell, when drug level is low, according to the interaction of JD6 on liposome and cell surface CD40L, can kill and wound more targetedly the target cell of expressing CD40L.In the time that drug level increases, the free drug that peptide modified methotrexate liposome and common methotrexate liposome discharge in culturing process is all enough to kill and wound a large amount of cells (cell survival rate is down to 30% left and right), 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 by the liposome drug-loading system of JD6 target mediation preparation, just likely use medicine still less to reach same result for the treatment of.
(2) the treatment checking of the molecular targeted peptide modified methotrexate liposome of CD40L to EAE mouse
1, the foundation of EAE mouse model, comprises the steps:
1) polypeptide myelin oligodendrocyte glycoprotein 35-55 is dissolved in PBS, and concentration is 2mg/ml; PT is dissolved in PBS, and concentration is 2 μ g/ml;
2) go isopyknic MOG35-55 solution and CFA to mix, be prepared into emulsion in Three-way injector, respectively 100 μ l emulsions are injected near the inguinal lymph nodes of the C57/BL6 mouse left and right sides, timing is modeling the 0th day;
3) latter the 1st day of injection, carries out PT tail vein injection to mouse after immunity, every 100 μ l;
4) observe mouse invasion situation every day, mark to 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 are divided into one-sided hind leg seriously benumbs; 3 are divided into bilateral hind leg seriously benumbs; ; 3.5 are 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 morbidity score curve, calculate morbidity ratio, investigate modeling situation, as shown in Figure 8, as shown in Figure 8: when mouse occurs after the successful symptom of typical EAE modeling, timing is the 0th day after treatment, and it is carried out to drug treatment, investigates the targeted therapy effect of the peptide modified methotrexate liposome of JD6.The initial average mark of each group EAE mouse is all 2 points, and PBS group can be indicated in the situation without pharmacological agent as blank group, can see the prolongation along with the time, and the incidence of mouse is serious gradually, and disease score curve is the trend rising gradually.Using in common methotrexate liposome therapeutic group and can see, the morbidity of EAE mouse mark all lower than PBS control group, illustrates and after use methotrexate liposome is treated mouse, still has significant curative effect.The methotrexate liposome that JD6 is peptide modified and the curative effect comparison of common methotrexate liposome, can see that the result for the treatment of of the peptide modified methotrexate liposome of JD6 is all better than common methotrexate liposome.Illustrate because it has the effect of CD40L target, can specific target and suppress to cause in EAE mouse the CD40L high expression level T cell of excessive inflammation.Consider that the PEG on common methotrexate liposome also has some nonspecific lymphsystem Targeting Effects, the result for the treatment of of the peptide modified methotrexate liposome of JD6 still can significantly be better than common methotrexate liposome, that is to say in the body of animal disease model in experiment, the effect of the drug-loaded liposome target high expression level CD40L lymph T cell of JD6 mediation is strong, and the disease causing for CD40L high expression level has better therapeutic action.
2, the result for the treatment of verification method of the molecular targeted peptide modified methotrexate liposome of CD40L to EAE mouse, comprises the steps:
1) according to EAE mouse invasion curve, in the time of modeling the 18th day, 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, and every group of 10 mouse, respectively organize average mark and are all 2 points, and one group of methotrexate liposome of modifying to JD6 is given for one group and had the common methotrexate liposome of identical PEG percentage composition, and one group to physiological saline, are designated as administration the 1st day;
3) follow-up respectively administration in the 4th, 8,12,15,18 days;
4) within every 3 days, give the scoring of EAE mouse, 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 points) respectively at the 10th day for every group, the PBMC in its blood of separation and purification, to its CD3, CD40L molecule carries out mark, and FACS detects various cellular component changing conditions.As shown in Figure 9, as shown in Figure 9: in peripheral blood lymphocyte, 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.Can see that normal mouse is about 0.01% in peripheral blood CD40L positive cell rate, and in EAE model mice (2 points of disease average marks) peripheral blood, CD40L positive cell ratio is higher, is 0.4%, exceeds very many than normal value.When using methotrexate liposome mouse to be carried out after 3 treatments, to treating mid-term, can see that significantly changing appears in the level of CD40L positive cell in Mouse Peripheral Blood Lymphocyte.Methotrexate liposome and common methotrexate liposome that JD6 modifies have all reduced 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 by JD6 target and mediate, the methotrexate liposome after peptide modified is better than non-targeted liposome to the fragmentation effect of CD40L high expression level T cell, and this advantage is just embodying clearly in mid-term for the treatment of.The drug-loaded liposome of CD40L target prepared by the method can, at the CD40L high expressing cell that suppresses to cause 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 in to 15mL centrifuge tube lower floor, 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, the acceleration of whizzer and deceleration are made as to 3, make it slowly accelerate and slow down, improve separating effect;
3) after centrifugal end, can on 1640 substratum and lymphocyte separation medium interface, see the white endless belt of clear densification, be buffy coat.With this confluent monolayer cells of aseptic straw sucking-off, then add 10ml1640 substratum, mix washing;
4) centrifugal 10 minutes of room temperature 300g, abandons supernatant, cell is resuspended in 1640 substratum to cell counting;
5) collect various cells to be measured, the FACS damping fluid re-suspended cell of precooling on ice, uses centrifuging washed twice, adjusts cell concn to 10
6individual/100 μ l;
6) in the cell in mouse source, add fluorescently-labeled CD3, CD40L antibody, mixes, and 4 DEG C of lucifuges are hatched 30 minutes, the FACS washed twice of precooling, and 1% paraformaldehyde stationary liquid is resuspended, adjusts concentration to by 10
6individual/1ml;
7) FACS detects the expression degree of corresponding fluorescently-labeled each molecule in FL-1/FL-2 passage.
In sum, of the present invention have is connected with DSPEPEG material with the polypeptide that the cell-specific of expressing CD40L molecule is combined after, be assembled on liposome, the liposome of this target polypeptide modification in vitro can well with the Cell binding of expression CD40L.Modify the liposome of parcel methotrexate by polypeptide DSPE-PEG connecting material, the activity of more effective inhibition CD40L positive cell in vitro in experimental autoimmune encephalomyelitis animal model, shows result for the treatment of simultaneously.
Claims (6)
1. a ligand polypeptide of being combined with CD40L protein-specific, is characterized in that, the sequence of described ligand polypeptide is as shown in SEQ ID NO.1.
2. a ligand polypeptide of being combined with CD40L protein-specific, it is characterized in that, described ligand polypeptide comprises sequence shown in SEQ ID NO.1, and adds linking group or connection aminoacid sequence at its C-terminal and/or N-terminal, and the sequence of described ligand polypeptide is as shown in SEQ ID NO.2.
3. a ligand polypeptide as claimed in claim 1 is in the purposes of preparing in delivery system.
4. a ligand polypeptide as claimed in claim 2 is in the purposes of preparing in delivery system.
5. a delivery system, is characterized in that, this system comprises: ligand polypeptide, drug-loading system, at least one active substance of being combined with CD40L protein-specific;
Described ligand polypeptide is connected to following arbitrary body structure surface: nanoparticle, polymkeric substance, liposome, vesica, solid lipid particulate, micella, carbon nanotube, organoid, lipoprotein;
The sequence of described ligand polypeptide is as shown in SEQ ID NO.1 or SEQ ID NO.2;
Described active substance is antitumor drug, antimetabolite, spindle body toxic alkaloid, topoisomerase enzyme inhibitor, photosensitizers, kinase inhibitor, microbiotic, immunosuppressor, antiparasitic compound, for the diagnostic reagent of ultrasonic contrast, pneumoradiography, goal gene, or the carrier for expression of eukaryon DNA that contains goal gene.
6. delivery system as claimed in claim 5, is characterized in that,
Described microbiotic is cytotoxic antibiotics;
Or described antitumor drug is antitumor antibiotics;
Or described goal gene is the combination of antisense oncogene, cytokine gene or aforementioned 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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| CN101855237A (en) * | 2007-11-05 | 2010-10-06 | 上海交通大学 | Peptide ligand directed drug delivery |
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