CN101536982A - Trastuzumab-modified toxin protein-coated PEG immune liposome and preparation and application thereof - Google Patents
Trastuzumab-modified toxin protein-coated PEG immune liposome and preparation and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of medicine. The traditional tumor treatment methods, such as radiation treatment, chemical treatment, biotic factor treatment, and the like are lack of targeting, and the immunotoxin treatment has the problem of sensibility of the nonspecific toxicity and the immunogenicity of toxic proteins, etc. The humanized McAb Trastuzumab-modified toxin protein-coated PEG immune liposome has the structure: an antibody is connected to the surface of lipidosome and can be combined with HER 2 antigen on the surface of a tumor cell so as to generate endocytosis; and the toxin proteins are coated in the lipidosome, and the sensibility of the nonspecific toxicity and the immunogenicity of the toxic proteins can be reduced. The invention also provides the preparation and the application of the lipidosome. Proved by the result of the experiment of killing the tumor cells in vitro, the biological targeting is favorable, and the function of killing the targeted tumor cells in vitro is outstanding; and the lipidosome has low toxic effect on normal cells.
Description
Technical field
The present invention relates to medical technical field, specifically, relate to a kind of PEGization immunoliposome of humanization monoclonal antibody trastuzumab modified packaged toxin protein, and preparation and application.
Background technology
Traditional tumor therapeuticing method comprises: radiotherapy, chemotherapy, biotic factor treatment etc.But these Therapeutic Method are not specifically at tumor cell, promptly lack targeting.Medicine also produces extensive toxicity to normal tissue cell in killing tumor cell.Cause the drug utilization degree limited, side effect is big, and using dosage is limited.Therefore, need a kind of effective targeting drug delivery system to make medicine arrive tumor area.This system not only can improve the concentration of medicine at tumor area, killing tumor cell more effectively, and can reduce infringement to normal cell, tissue.
Cell surface has receptor, in theory, can combine with ligands specific.Because this binding specificity is very strong, we can utilize the guide effect of part, and to purpose tissue or cell, the performance drug effect realizes targeted therapy with the medicine directional guide.Trastuzumab is the antigenic Humanized monoclonal antibodies of a kind of anti-HER2, can discern and be incorporated into human epidermal growth factor acceptor-(HER2) (referring to patent JP3502885, US5677171, the WO2004008099) of cell surface specifically.Existing bibliographical information HER2 high expressed in many solid tumors, as knot endometrial carcinoma, pulmonary carcinoma, rectal cancer, gastric cancer, cancer of pancreas, ovarian cancer, bladder cancer etc. (referring to document Hynes NE, Stern DF.The biology of erbB-2/neu/HER-2and its role incancer.Biochem Biophys Acta 1198,1994,165-184.).
At present, the common method of bioprotein class Drug therapy tumor is the immunotoxin treatment.Immunotoxin is a kind of fusion rotein that is combined to form through technique for gene engineering by toxin protein and targeting part (antibody or antibody fragment).The mechanism of its killing tumor cell is by the receptor on the targeting part specific bond cell, immunotoxin by cell endocytic after, toxin protein impels apoptosis of tumor cells.Yet the effect of immunotoxin clinical treatment tumour does not reach gratifying degree, and most immunotoxins stop at clinical I, II test.Main cause is as follows: at first, the non-specific liver toxicity of toxin protein and blood vessel toxicity can produce vascular leak syndrome (VLS), cause therapeutic dose to be restricted thus.Secondly, because immunotoxin has very strong immunogenicity, can occur the antitoxin neutrality antibody rapidly in the human body after the administration, thereby limit its clinical repeated application.Therefore, when the preparation immunotoxin, how reducing the toxin protein non-specific toxicity and reducing its immunogenic sensitivity also is problem demanding prompt solution.In addition, protein medicaments has the advantages that molecular weight reaches the space structure complexity greatly, very easily is subjected to complex physical environmental effect (the particularly effect of a large amount of enzyme materials) in the drug delivery process, and proteic structure is damaged, and causes its loss of activity.By the preparation nanoparticle, albumen is wrapped up and can effectively address these problems.
It is CN200810035706.6 that the applicant has submitted number of patent application on April 8th, 2008, and denomination of invention is the Chinese invention patent application of " targeted nano granule of humanization monoclonal antibody trastuzumab modified packaged toxin protein and preparation thereof and application ".
Follow-up study is found, by selecting matrix material with excellent biological compatibility, toxin protein wrapped up be prepared into liposome, can form initiatively targeting, and also can effectively solve toxin protein non-specific toxicity, immunogenic sensitivity, and the problems such as loss of activity of toxin protein.U.S. FDA approved Evacet preparation is used for the therapeutic alliance of multiple myeloma, and there have multiple antitumor Liposomal formulation also to be applied to be clinical.
Summary of the invention
The object of the invention provides a kind of PEGization immunoliposome of packaged toxin protein of tumor cell of selectively targeted HER2 high expressed, the non-specific toxicity of toxin protein of parcel and immunogenic sensitivity are reduced and keeps that toxin protein structure in application process is not damaged, activity can not lost.
Another object of the present invention provides the preparation of above-mentioned immunoliposome.
Another object of the present invention provides the application of above-mentioned immunoliposome.
The drug-loaded liposome of target tumor cell, its structure should comprise (1) part usually, can discern and in conjunction with the tumor cell surface specific receptor, easily by cell endocytic; (2) liposome of the matrix material of biocompatibility preparation, parcel and delivery antitumor drug; (3) linking group connects described part and liposome.
The present invention connects humanization monoclonal antibody trastuzumab on the nanoparticle surface, and this antibody can combine and take place cell endocytic with tumor cell surface HER2 antigen.By the guiding function of antibody, the immunoliposome of PEGization can be applied to the tumor of all HER2 overexpressions in principle.
The present invention selects matrix material with excellent biological compatibility: i.e. EPC (eggphosphatidylcholine, Ovum Gallus domesticus Flavus lecithin), CHOL (cholesterol, cholesterol), mPEG
2000-DSPE (1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine[methoxy (polyethyleneglycol)-2000], methoxy poly (ethylene glycol) 2000-distearyl phosphoglyceride acyl ethanolamine), and PDP-mPEG
2000-DSPE (1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine[pyridyldit hioproprionate methoxy (polyethyleneglycol)-2000], pyridine dithio propionic acid amide .-methoxy poly (ethylene glycol) 2000-distearyl phosphoglyceride acyl ethanolamine) as the biocompatibility matrix material for preparing liposome.
The invention provides a kind of PEGization immunoliposome of humanization monoclonal antibody trastuzumab modified packaged toxin protein, comprise following structure:
---part, be humanization monoclonal antibody trastuzumab, the human epidermal growth factor acceptor of tumor cell surface-(HER2) can optionally be discerned and be incorporated into to this part;
---the liposome of biocompatibility matrix material preparation, parcel and delivery antitumor toxin protein medicine in the liposome, described biocompatibility matrix material is EPC, CHOL, mPEG
2000-DSPE and PDP-mPEG
2000-DSPE is 2: 1: 0.08 with mol ratio: 0.02 mixes; And
---linking group, be sulfydryl and SMPB (N-succinimidyl-4-(p-maleimidophenyl)-butyrate, N-succinimido-4-is to the maleimide phenyl butyrate), connect described part and the surperficial liposome that has sulfydryl.
In addition, the size of liposome also can influence the targeting of medicine, if particle diameter is excessive, then is easy to be engulfed by reticuloendothelial system (RES) in blood circulation, causes medicine to be removed rapidly, can't realize the target tumor cell.The particle diameter preferable range of the drug-loaded liposome of selectively targeted tumor cell of the present invention is the 100-200 nanometer, can effectively reduce engulfing of RES, improves the targeting of pharmaceutical preparation.
Parcel and delivery antitumor toxin protein medicine are water miscible antitumor toxin proteins in the above-mentioned liposome, specifically are meant: bacteriotoxin (as diphtheria toxin, diphtherotoxin DT and pseudomonas extracellular toxin PE) or phytotoxin (as Ricin Ricin) and their mutant.
The present invention also provides the preparation method of the PEGization immunoliposome of above-mentioned humanization monoclonal antibody trastuzumab modified packaged toxin protein, may further comprise the steps:
(A) adopt film dispersion method, the PEGization liposome that contains the PDP group of preparation parcel and delivery antitumor toxin protein, the material of preparation liposome is EPC, CHOL, mPEG
2000-DSPE, PDP-mPEG
2000-DSPE is 2: 1: 0.08 with mol ratio: 0.02 mixes;
(B) adopt the chemical coupling method, the liposome of humanization monoclonal antibody trastuzumab with parcel and delivery antitumor toxin protein is connected, linking group is sulfydryl and SMPB.
Concrete steps are as shown in Figure 2:
(A) contain the preparation of the PEGization liposome of PDP group
With EPC, CHOL, mPEG
2000-DSPE, PDP-mPEG
2000-DSPE is 2: 1: 0.08 with mol ratio: 0.02, be dissolved in an amount of chloroformic solution, and make lipid membrane.With prepared lipid membrane with the abundant aquation of antitumor toxin protein solution, make liposome turbid liquor, cross film with the thin film extruder again, cross the antitumor toxin protein that post will not be wrapped then and remove, will obtain liposome turbid liquor after the sample concentration of collecting.
(B) modification of antibody and modify after being connected of antibody and liposome
According to document Allen, T.M., Brandeis, E., Hansen, C.B., et al.A new strategy forattachment of antibodies to sterically stabilized liposomes resulting in efficienttargeting to cancer cells.Biochim.Biophys.Acta., 1995; 1237:99-108. disclosed chemical coupling method is connected antibody with liposome.Earlier with antibody with SMPB (N-succinimidyl-4-(p-maleimidophenyl)-butyrate, N-succinimido-4-is to the maleimide phenyl butyrate) modify and to make MPB-Ab, then at DTT (dl-dithiothreitol, dithiothreitol, DTT) under the effect reduction of PDP group is prepared into the liposome that contains SH (sulfydryl), lip-deep sulfydryl of drug-loaded liposome and MPB-Ab reaction, part is connected with drug-loaded liposome, makes the PEGization immunoliposome.
Above-mentioned coupling reaction when antibody is connected with liposome, adopts sulfydryl and N-succinimido-4-to maleimide phenyl butyrate (SMPB).
The temperature of above-mentioned coupling reaction is advisable with 10-30 ℃.
Immunoliposome of the present invention is compared with simple PEGization liposome, with PDP-mPEG
2000-DSPE is the immunoliposome that is prepared into after lipid feedstocks is modified, and can form initiatively targeting.The present invention is wrapped in toxin protein in the liposome, and the chance that toxin protein contacts with normal structure will reduce greatly, thereby reduces the toxin protein non-specific toxicity; Parcel can reduce the contacting of neutrality antibody of toxin protein and body generation again simultaneously, can further reduce its immunogenic sensitivity.
The present invention also provides the application of PEGization immunoliposome in the preparation antitumor drug of above-mentioned humanization monoclonal antibody trastuzumab modified packaged toxin protein.The tumor cell of particularly selectively targeted antigen HER2 high expressed is as knot endometrial carcinoma, pulmonary carcinoma, rectal cancer, gastric cancer, cancer of pancreas, ovarian cancer, transitional cell bladder carcinoma cell line etc.Liposome of the present invention is tested through external killing tumor cell, and the result proves that the biological target tropism is good, and external to kill and wound the effect of target tumor cell remarkable; Very little to normal cytotoxic effect simultaneously.
Description of drawings
Fig. 1 is the particle size distribution figure of the PEGization immunoliposome of humanization monoclonal antibody trastuzumab modified parcel PE38KDEL of the present invention.
Fig. 2 is the preparation sketch map of the PEGization immunoliposome of humanization monoclonal antibody trastuzumab modified parcel PE38KDEL of the present invention.
Fig. 3 is the PEGization liposome cumulative in vitro release～time plot of parcel PE38KDEL of the present invention.
Fig. 4 is PEGization immunoliposome cell in vitro poison lab diagram, the wherein A of humanization monoclonal antibody trastuzumab modified parcel PE38KDEL of the present invention: each preparation kills and wounds the SK-BR3 cell of HER2 antigen high expressed; B: each preparation is to HER2 antigen killing and wounding than low MDA-MB-231 cell of expressing; C: each preparation kills and wounds the low MCF-7 cell of expressing of HER2 antigen.
The specific embodiment
Describe the present invention below in conjunction with drawings and Examples, but enforcement of the present invention is not limited only to this.
Embodiment 1: antitumor toxin protein PE38KDEL separation and purification
PE38 is that (molecular weight is 38KD to pseudomonas extracellular toxin in the bacteriotoxin for pseudomonas extoxin, PE) a kind of, and PE38KDEL is preferred PE38 mutant, has the little advantage of non-specific toxicity (the The 2nd Army Medical College institute of oncology provides).
Reference literature Song S, Xue J, Fan K, et al.Preparation and characterization of fusionprotein truncated Pseudomonas Exotoxin A (PE38KDEL) in Escherichia coli.ProteinExpr Purif 2005; 44:52-57. method, at first make up pET (Novagen) expression vector of PE38KDEL, change over to then engineering bacteria BL21 (DE3) (Novagen) in, utilize derivant IPTG (isopropy-β-D-thiogalactoside, isopropyl-) abduction delivering to go out the PE38KDEL toxin protein.Then, obtain the PE38KDEL of solubility expression in antibacterial by the method for nickel post affinity chromatograph.At last by the external protein translation of rabbit reticulocyte lysate system, cell killing is tested the activity that confirms PE38KDEL, and (document Gao J sees reference, Kou G, Wang H, et al.PE38KDEL-loaded anti-HER2 nanoparticles inhibit breast tumor progression withreduced toxicity and immunogenicity.Breast Cancer Res.Treat., doi:10.1007/s 10549-008-0043-0.).
Embodiment 2: the preparation of the PEGization liposome of parcel antitumor toxin protein PE38KDEL
With the concentration of the aqueous solution of toxin protein PE38KDEL after MicroBCA method (Pierce company) the micrometric measurement separation and purification, concentration range is advisable at 2-10mg/ml.With EPC, CHOL, mPEG
2000-DSPE (2: 1: 0.08, mol ratio) is dissolved in the chloroformic solution of 2～3ml, and this solution is joined in the pyriform flask, and inflated with nitrogen three times is flung to organic solvent with Rotary Evaporators then fully under vacuum condition.With the PE38KDEL solution abundant aquation of prepared lipid membrane with 2mg/ml, make liposome turbid liquor, cross the polycarbonate membrane of 800nm, 400nm, 200nm more respectively with the thin film extruder, be that eluent is crossed the PE38KDEL that Sepharose CL-4B post will not be wrapped and removed with PBS (pH 7.4) then, with the sample collected with 1000, the ultra-filtration centrifuge tube of 000Da obtains the liposome turbid liquor that phospholipid concentration is 20 μ mol/ml at 2000rpm after concentrating under 6 ℃ of conditions.
Particle size distribution range is 100-200nm, as shown in Figure 1.
Embodiment 3: the antibody after the modification of antibody and the modification is connected with liposome
(1) preparation of MPB-Ab (maleimidophenylbutyrate-HER2)
Ab (antibody trastuzumab, trade name Herceptin buy the company in Genentech) is dissolved in (pH 7.4 for 25mM HEPES, 140mM NaCl) in the HEPES buffer, is prepared into the Ab solution of 10mg/ml.SMPB (N-succinimidyl-4-(p-maleimidophenyl)-butyrate with 25mM, N-succinimido-4-is to the maleimide phenyl butyrate) be dissolved in DMF (dimethyl formamide solution), join lentamente then (mol ratio of SMPB and Ab is 20: 1) in the above-mentioned Ab solution, reaction is 30 minutes under the room temperature.Then with HEPES and MES (25mM HEPES, 25mM MES, 140mM NaCl, pH 6.7) cross Sephadex G50 post for eluent unconjugated SMPB is removed, with the sample collected with 3, the ultra-filtration centrifuge tube of 000Da is at 2000rpm, obtains the MPB-Ab solution of 10mg/ml after concentrating under 6 ℃ of conditions, is equipped with MPB-BSA (bovine serum albumin) in contrast with legal system.
(2) combination of antibody
Being prepared as follows of the immunoliposome of PEGization.At first with EPC/CHOL/mPEG
2000-DSPE/PDP-PEG
2000The liposome prescription of-DSPE (2: 1: 0.08: 0.02, mol ratio) makes lipid membrane (the total molal quantity of PEG be phospholipid 5%).Then with after the abundant aquation of PE38KDEL solution of prepared lipid membrane with 2mg/ml, at ambient temperature, with the DTT (dithiothreitol, DTT) of 20mM with the pyridine disulfide group reduction of liposome outside 30 minutes.Cross the SephadexG50 post with HEPES and MES (140mM NaCl, pH 6.7 for 25mM HEPES, 25mM MES) for eluent then and remove DTT.Liposome of sulfhydrylation (20mM phospholipid) and MPB-Ab be overnight incubation at room temperature, and the mol ratio of Ab and phospholipid is 1: 1330.With PBS (pH 7.4) is that eluent is crossed Sepharose CL-4B post with the liposome that makes and removed unconjugated MPB-Ab.The unconjugated amount of MPB-Ab is measured by the MicroBCA method.The PEGization immunoliposome that makes is with 1000, and the ultra-filtration centrifuge tube of 000Da is at 2000rpm, be concentrated into 54mM under 6 ℃ of conditions after, fill N
2, airtight preservation or lyophilization get final product.
Preparation method of the present invention sees Fig. 2 for details.
Embodiment 4: the extracorporeal releasing experiment of the PEGization immunoliposome of the humanization monoclonal antibody trastuzumab modified parcel PE38KDEL of invention
Liposome 10.02～10.07mg that precision takes by weighing the parcel PE38KDEL of 4 different batches places 1000 respectively, in the 20ml ultra-filtration centrifuge tube of 000Da, the PBS buffer (containing 0.02% sodium azide as antibacterial) that adds an amount of pH7.4 is release medium, place the water bath with thermostatic control shaking table, under 100rpm hunting speed, 37 ℃ ± 0.5 ℃ temperature conditions, wrap up the release in vitro degree of PE38KDEL liposome and measure.Respectively 1,2,4,6,9,12 and 24h take out ultra-filtration centrifuge tube, in at 2000rpm, centrifugal 20min under 4 ℃ of conditions, clear liquid under the sucking-off, with release medium the release medium in the ultra-filtration centrifuge tube is supplied the content of PE38KDEL in the clear liquid under measuring with the MicroBCA method more then.Do not adding mPEG
2000Under the situation of-DSPE, with the conventional liposome of embodiment 2 same procedure preparations in contrast.
As shown in table 1, PE-Liposomes (the PEGization liposome of packaged toxin protein) and PE-HER-Liposomes (the PEGization immunoliposome of the packaged toxin protein that Ab modifies) no significant difference aspect envelop rate and drug loading (P＞0.05), the quantitative of PE38KDEL release in vitro disturbed in the combination of Ab in addition, therefore we use PE-Liposomes, rather than PE-HER-Liposomes carries out the extracorporeal releasing experiment of PE38KDEL.
The feature of the PEGization liposome of table 1 parcel PE38KDEL
aAll?data?are?expressed?as?the?mean±SD(n＝3).
bP＞0.05；there?was?no?significant?difference?between?PE-liposomes?and?PE-HER-liposomes?in?the?EE?and?drugloading?using?Student’s?unpaired?t?test.
The result: conventional liposome is followed successively by at the cumulative release percentage ratio of each sample point: 25.14 ± 3.41,44.72 ± 4.99,56.28 ± 6.36,69.57 ± 5.80,80.06 ± 5.35,86.97 ± 7.13,99.23 ± 0.52 (n=4); The PEGization liposome is followed successively by at the cumulative release percentage ratio of each sample point: 5.18 ± 0.62,10.27 ± 1.84,13.65 ± 3.11,16.41 ± 3.46, and 19.50 ± 4.29,23.14 ± 4.88,33.07 ± 5.73 (n=4) sees Fig. 3 for details.The PEGization immunoliposome preparation of prepared as seen from the figure parcel PE38KDEL can satisfy the needs as effective delivery system.
Embodiment 5: the external killing tumor cell experiment of the PEGization immunoliposome of the humanization monoclonal antibody trastuzumab modified parcel PE38KDEL of invention
Cytotoxicity experiment is by (the Promega of manufacturer, Madison, WI) experimental design is measured (Chen with the reagent for determination of non-radioactive cell proliferation box in cell titration 96 holes, H., Gao, J., Lu, Y., et al.Preparation and characterization of PE38KDEL-loaded anti-HER2 nanoparticles fortargeted cancer therapy.J.Control.Release, 2008; 128:209-216.).Under the condition of PEGization liposome (embodiment 2 preparations), the immunoliposome (embodiment 1-3 preparation) of PEGization, free PE38KDEL (embodiment 1 preparation) or each blank liposome, with breast cancer cell (1 * 10
4) in 37 ℃, CO
2Couveuse was hatched two days.The mensuration concentration from 1 to 20 of PE38KDEL in this method, 000pM (equaling total lipid concentration of 0.1mg/ml).To not have the liposome of blank PEGization of PE38KDEL and immunoliposome the hatching under the highest total lipid concentration (0.1mg/ml) condition of PEGization to measure cytotoxicity over two days, then 20 μ l MTS/PMS solution will be added in each hole.Under 37 ℃ of conditions, hatched 2 hours, measure the trap of sample in each hole with microplate reader at the 490nm wavelength.
The result shows, the immunoliposome of PEGization is better than the liposome and the free PE38KDEL of PEGization to killing and wounding of SK-BR3, sees Fig. 4 for details; There are not the liposome of blank PEGization of PE38KDEL and immunoliposome no cytotoxicity under the highest total lipid concentration (0.1mg/ml) condition of PEGization.
Claims (8)
1, the PEGization immunoliposome of humanization monoclonal antibody trastuzumab modified packaged toxin protein comprises following structure, and particle size range is the 100-200 nanometer:
Part is humanization monoclonal antibody trastuzumab;
The liposome of parcel and delivery antitumor toxin protein, the material of preparation liposome is EPC, CHOL, mPEG
2000-DSPE and PDP-mPEG
2000-DSPE is 2: 1: 0.08 with mol ratio: 0.02 mixes; And
Linking group is sulfydryl and SMPB, connects described part and the surperficial liposome that has sulfydryl.
2, the PEGization immunoliposome of humanization monoclonal antibody trastuzumab modified packaged toxin protein according to claim 1 is characterized in that antitumor toxin protein wherein is bacteriotoxin or phytotoxin, and their mutant.
3, the PEGization immunoliposome of humanization monoclonal antibody trastuzumab modified packaged toxin protein according to claim 2 is characterized in that bacteriotoxin wherein is diphtheria toxin, diphtherotoxin or pseudomonas extracellular toxin.
4, the PEGization immunoliposome of humanization monoclonal antibody trastuzumab modified packaged toxin protein according to claim 2 is characterized in that phytotoxin wherein is a Ricin.
5, a kind of preparation method of PEGization immunoliposome of humanization monoclonal antibody trastuzumab modified packaged toxin protein as claimed in claim 1 may further comprise the steps:
(A) adopt film dispersion method, the PEGization liposome that contains the PDP group of preparation parcel and delivery antitumor toxin protein, the material of preparation liposome is EPC, CHOL, mPEG
2000-DSPE, PDP-mPEG
2000-DSPE is 2: 1: 0.08 with mol ratio: 0.02 mixes;
(B) adopt the chemical coupling method, the liposome of humanization monoclonal antibody trastuzumab with parcel and delivery antitumor toxin protein is connected, linking group is sulfydryl and SMPB.
6, preparation method according to claim 5 is characterized in that concrete steps are as follows:
(A) with EPC, CHOL, mPEG
2000-DSPE, PDP-mPEG
2000-DSPE is 2: 1: 0.08 with mol ratio: 0.02 is dissolved in chloroformic solution, make lipid membrane, with prepared lipid membrane with the abundant aquation of antitumor toxin protein solution, make liposome turbid liquor, cross film with the thin film extruder again, cross the antitumor toxin protein that post will not be wrapped then and remove, concentrate;
(B) humanization monoclonal antibody trastuzumab is made MPB-Ab with the SMPB modification, under the effect of DTT the reduction of PDP group is prepared into the liposome that contains sulfydryl then, sulfydryl on the surface of liposome and MPB-Ab reaction are connected part with liposome; Reaction temperature is 10-30 ℃.
7, the application of a kind of PEGization immunoliposome of humanization monoclonal antibody trastuzumab modified packaged toxin protein as claimed in claim 1 in the preparation antitumor drug.
8, the application of the PEGization immunoliposome of humanization monoclonal antibody trastuzumab modified packaged toxin protein according to claim 7 in the preparation antitumor drug, tumor wherein is carcinoma of endometrium, pulmonary carcinoma, rectal cancer, gastric cancer, cancer of pancreas, ovarian cancer or bladder cancer.
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| CN106546705A (en) * | 2015-09-21 | 2017-03-29 | 上海复旦张江生物医药股份有限公司 | A kind of method of testing of liposome medicament release in vitro |
| CN105651987A (en) * | 2016-01-29 | 2016-06-08 | 苏州联辰生物技术有限公司 | Method for manufacturing novel nano beam concentration material |
| CN109862883A (en) * | 2016-10-21 | 2019-06-07 | 生物权威（英国）有限公司 | Cytotoxicity particle |
| CN109498817A (en) * | 2017-09-14 | 2019-03-22 | 上海交通大学 | A kind of many cells target liposomes |
| CN113893352A (en) * | 2021-09-30 | 2022-01-07 | 清华大学深圳国际研究生院 | JO protein anchored drug-loaded immune cell and preparation method and application thereof |
| CN114404614A (en) * | 2022-01-28 | 2022-04-29 | 上海交通大学 | Immune liposome targeting pseudomonas aeruginosa as well as preparation method and application thereof |
| CN114404614B (en) * | 2022-01-28 | 2024-04-23 | 上海交通大学 | Immune liposome targeting pseudomonas aeruginosa and preparation method and application thereof |
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