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In the study, we developed a novel formulation, CD123 mono-antibody (mAb) modified tanshinone ⅡA loaded immunoliposome (CD123-TanⅡA-ILP) to achieve the targeted drug delivery for leukemia cells. Orthogonal test was used to optimize liposome preparation, and the TanⅡA-loaded PEGylated liposomes (TanⅡA-LP) of S100PC-Chol-(mPEG2000-DSPE)-TanⅡA at 19∶5∶1∶1 molar ratio were prepared by the thin film hydration-probe ultrasonic method. A post-insertion method was applied to prepare CD123-TanⅡA-ILP via thiolated mAb conjugated to the terminal of maleimide-PEG2000-DSPE. The cellular uptake assay was measured by flow cytometry, and the inhibitory effect of CD123-TanⅡA-ILP on NB4 cells proliferation was tested by using MTT assay. The results of cellular uptake assay showed that CD123-ILP could significantly increase the drug uptake of NB4 cells as compared with free drugs and LP. The IC₅₀ values at 48 h incubation were 20.87, 11.71, 7.17 μmol•L⁻¹ respectively for TanⅡA，TanⅡA-LP and CD123-TanⅡA-ILP. CD123-ILP demonstrated a potential and promising targeted drug delivery strategy for acute myelogenous leukemia (AML) treatment.
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In this study,norcanthridin(NCTD)-encapsulated liposomes were modified with a novel murine anti-human CD19 monoclonal antibody 2E8(2E8-NCTD-liposomes)and the targeting efficiency and specific cytotoxicity of 2E8-NCTD-liposomes to CD19+leukemia cells were evaluated.BALB/c mice were injected with 2E8 hybridoma cells to obtain 2E8 monoclonal antibody(mAb).NCTD-liposomes were prepared by using film dispersion method.2E8 mAbs were linked to NCTD-liposomes using post-incorporation technology.Flow cytometry showed that the targeting efficiency of purified 2E8 mAbs on CDI9+Nalm-6 cells was 99.93%.The purified 2E8 mAbs were conjugated with NCTD-liposomes to prepare 2E8-NCTD-liposomes whose targeting efficiency on CD19+Nalm-6 was also 95.82%.The average size of 2E8-NCTD-liposomes was 118.32 nm in diameter.HPLC showed that the encapsulation efficiency of NCTD was 46.51%.When the molar ratio of 2E8/MaI-PEG2000-DSPE reached 1:50,we obtained the liposomes with 9 2E8 molecules per liposome.The targeting efficiency of 2E8-NCTD-liposomes on CD19+leukemia cells was significantly higher than that on CD19-leukemia cells.Similarly,the targeting efficiency of the immunoliposomes was also higher than that of the NCTD-liposomes on CD 19+leukemia cells.Those results were consistent with those observed by laser scanning confocal microscopy.3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay demonstrated that 2E8-NCTD-liposomes specifically killed Nalm-6 cells in a dose-and time-dependent manner.The viability of Nalm-6 cells treated by 2E8-NCTD-liposomes was significantly lower than that of Molt-3cells and it was also significantly lower than that of Nalm-6 cells treated with the same concentration of NCTD-liposomes or free NCTD.We are led to concluded that 2E8 antigen can serve as a specific targeting molecule of B lineage hematopoietic malignancies for liposome targeting,and 2E8-NCTD-liposomes can be used as a new and effective means for the treatment of B lineage hematopoietic malignancies.
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AIM: To prepare urokinase-loaded immunoliposomes, with anti-D-dimer mouse monoclonal antibody that can selectively target to thrombotic site and test the thrombolytic effect in the rabbit model of acute pulmonary embolism (PE) in early phase. METHODS: 40 New Zealand white rabbits were randomly divided into five groups: TBS group (TBS buffer, negative control group), urokinase (UK) group (150 000 IU/kg UK, positive control group), Lip group (Lip-UK, containing 50 000 IU/kg UK), Ab group (Ab-Lip-UK, containing 50 000 IU/kg UK) and 2 Ab group (2 Ab-Lip-UK, containing 50 000 IU/kg UK but double DDmAb of Ab group). PE model was established. Five minutes later, five solutions (TBS, UK, Lip-UK, Ab-Lip-UK, 2 Ab-Lip-UK) were transfused through femoral vein respectively. Right ventricular systolic pressure (RVSP) and right ventricular diastolic pressure (RVDP) were estimated within one hour. The pathological changes of lung, heart, liver and kidney were all observed. RESULTS: RVSP in TBS group had no significant changes within one hour after administration. However, RVSP had respective notable decline at 30 min, 40 min, 30 min, 20 min in UK, Lip, Ab and 2 Ab group, respectively. The means of residual emboli were as follows: 4.0±0 in TBS group, 2.4±0.9 in UK group, 3.1±0.6 in Lip group, 2.4±0.9 in Ab group and 1.9±0.6 in 2 Ab group. The lungs in each group showed scattering local congestion, effusion and swelling on the surface. Obvious hemorrhage of heart, kidney and liver was found with HE staining in UK group, no pathologic change was observed in other groups. CONCLUSION: 2 Ab-Lip-UK with early thrombolytic effect and security may be an ideal thrombolytic agent for treating pulmonary embolism.
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objective To observe whether the killing effect on HCC SMMC-7721 cell of the antihepatocellular carcinoma scFv reconstructed by pharmacy was enhanced or not.Methods Prokarycytic expression vector containing PET32a-RC-RNase was induced to express by IPTG.The inclusion body purified and Western-blotting was used.PC.CHOL and CHS was added in chloroform.Dry membrane was formed after chloroform was removed.RC-RNase protein solution was added to dissolute the membrane.Then pass the solution over a Sephadex G-50 column after ultrasound and filtrated to detect the encapsulation efficiency of the liposome.The solution reacted in EDC.SSNHS and MES for 30 minutes.Then add hdscFv to the solution in 4 ℃ over night.MTT method was used to detect the killing effect on HCC cell of immunoliposome RC-RNase,immunotoxin RC-RNase and liposome RC-RNase in vitro.Resuits The killing effect on HCC cell of immunoliposome RC-RNase is the best.but that of Iiposome RC-RNase is the worst.The respective JC50 are:3.28μg/ml,22.44μg/ml and 98.26μg/ml.Conclusion The anti-hepatocellular carcinoma scFv relomtructed by pharmacy can promote the killing effect on HCC cell and may have potential in the treatment of hepatocarcinoma.
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Objective:To search for a simple and rapid method of preparating the IL-2 conjugated immunoliposomes with high conjugate rate.Methods:The IL-2 conjugated immunoliposomes were prepared by Glutaraldhyde,Undecanal or Derivatization.All methods were analyzed and compared.The conjugate rate of IL-2 was determined by Bradford Assay.Centrifugal acceleration experiment served to prove the liposome stability.Results:IL-2 conjugated liposome by shiff’s base through Undecanal didn’t cause IL-2-self or liposme-self inter-cross link,and conjugate rate was high and stability was good.Conclution:Undecanal is suggested to prepare the IL-2 conjugated immunoliposomes for the anaphase cell experiment.
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Aim To prepare sterically stabilized liposomes modified with chimeric TNT-3 monoclonal antibody (chTNT-3) and investigate their immunoreactivity and in vitro targeting. Methods An endgroup functionalized polyethylene glycol-lipid derivative (pyridylthiopropionoylamino-polyethylene glycolhydrogenated soy phosphatidylethanolamine, PDP-PEG-HSPE ) was synthesized and incorporated to sterically stabilized liposomes. After mild thiolysis of the PDP groups by dithiothreitol, liposomes were covalently linked with maleimide-derivatized chTNT-3 and formed sterically stabilized immunoliposomes.Coupling efficiency, antibody density, size distribution, immunoreactivity of chTNT-3-modified sterically stabilized liposomes (chTNT-3-SLs) and specific binding properties of the chTNT-3-SLs to fixed Raji cells were determined, separately. Results Higher initial Ab/PDP-PEG-HSPE molar ratios resulted in higher antibody density on the surface of liposomes but lower coupling efficiency. The optimal coupling efficiency of 71% was obtained while antibody density in liposome was 106 μg antibody/μmol phospholipids (as initial antibody/PDP-PEG-HSPE = 1: 10). The chTNT-3-SLs had a narrow size distribution after extrusion and the mean size of this immunoliposomes was (115 ± 33) nm. The immunoreactivity of chTNT-3 can be preserved after efficient attachment of maleimide-derivatized chTNT-3 to the surface of liposomes. But calculated per antibody concentration, the immunoreactivity of chTNT-3-SLs would obviously decrease compared to that of chTNT-3 or chTNT-3 derivatives. Significantly higher binding of chTNT-3-SLs to fixed Raji cells directed by chTNT-3 was obtained compared to other preparations in serial dilutions (P＜0.01). Conclusion chTNT-3-SLs prepared by PDP-PEG-HSPE method remained most immunoreactivity of chTNT-3 and was able to bind nuclear antigens in vitro.
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Objective:To compare the killing effects on HCC SMMC-7721 cells of immunoliposome PE38、immunotoxin PE38 and liposome PE38. Methods:Dissolute PC,CHOL and CHS in chloroform in proportion.Dry membrane was formed after chloroform was removed.Add the protein solution of PE38 to dissolute the dry membrane.Then pass the solution over a Sephadex G-50 column after ultrasoned and filtrated to detect the encapsulation efficiency of the liposome.The solution reacted in EDC,SSNHS and MES for 30 minutes.Then add Ab to the solution in 4℃ over night.MTT method was used to detect the killing effects on HCC cells of immunoliposome PE38、immunotoxin PE38 and liposome PE38 in vitro. Results:The killing effects on HCC cells of immunoliposome PE38 is the best,and that of liposome PE38 is the worst.The respective IC_(50) s are:0.59 ?g/ml、6.04 ?g/ml and 92.07 ?g/ml. Conclusion:Immunoliposome PE38 may be a protential durg in the treatment of hepatocarinoma.
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Objective To prepare liposome-entrapped mitoxantrone (LEM)-GM-CSF and observe the cytotoxicity of HL-60/ADM cells treated with LEM-GM-CSF, LEM and dihydroxyanthraquinone (DHAQ) in vitro. Methods LEM was prepared by reverse phase evaporation (REV). High speed centrifugation was applied to separate LEM and dissociate DHAQ. Colorimetry was employed to determine encapsulation efficiency. The liposome structure and particle size were determined by transmission electron microscopy. GM-CSF was coupled to LEM by glutaraldehyde method. UV-spectrophotometric analysis was applied to measure the coupled efficiency. Flow cytometry was applied to determine the immunoconjugate retained efficiency. The cytotoxicity of HL60/ADM cells and interdiction efficiency of GM-CSF were investigated by MTT test. Results The encapsulation efficiency of LEM was 80%. Most liposomes were monolayer, and the particle size was 170-220 nm. Its coupled efficiency with GM-CSF was 42.3%, and the immunoconjugate retained efficiency was 74.6%. All LEM-GM-CSF, LEM and DHAQ had cytotoxicity on HL60/ADM, their cytotoxic power in decrement sequence: LEM-GM-CSF, LEM, DHAQ. After treated with LEM-GM-CSF, LEM and DHAQ for 24 h, the IC50 of HL-60/ADM was 8.73, 12.42, 27.31 ?g/ml respectively and for 48 h the IC50 were 0.62, 8.25, 12.44 ?g/ml. The inhibition rate increased in a dose-dependent manner. Conclusion The encapsulation efficiency, the coupled efficiency and the immunoconjugate retained efficiency of LEM-GM-CSF prepared by our method were satisfying. LEM-GM-CSF representing anti-leukaemia efficiency in vitro had cytotoxicity on HL60/ADM cells.
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Objective To evaluate the effect of albendazole immunoliposome (IL-Alb) against Echinococcus granulosus. Methods Mice infected with protoscolices of E.granulosus were divided into five groups. Four groups were treated with albendazole (Alb), albendazole liposome (L-Alb), albendazole sulfoxide liposome (L-Albso), and IL-Alb respectively at a dosage of 100 mg (Alb)/(kg?d)?5 d for 3 courses. The fifth group was established as control. The major criteria for evaluating the effects included a reduction rate of E.granulosus tissue wet weight, histopathological examination of the cysts by both light microscopy and electron-microscopy, and the content of albendazole-sulfoxide in cysts detected by HPLC. Results The efficacy of albendazole immunoliposome was significantly higher than that of albendazole liposome, and much higher than that of albendazole. The reduction rates of cyst tissue weight of IL-Alb group, L-Alb group and Alb group were 91^5%, 80^3%, 61^2% respectively as compared to control group; the concentration of Albso in cyst tissue of the above groups were 5^15 ?g/g, 2^18 ?g/g, 0^76 ?g/g respectively (P
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Objective:To prepare immunoliposomes encapsulating alkaline phosphatase and to evaluate the methods.Methods:The liposomes containing alkaline phosphatase were prepared by reverse phase evaporation or film dispersion.Liposomes and rabbit anti-human IgG were conjugated by glutaraldhyde or deriving antibody.Not only did the liposomes encapsulate alkaline phosphatase but also they had immune activity.All methods were analyzed and compared.Results:Many liposomes prepared by reverse phase evaporation had perfect shape and high bioactivity of alkaline phosphatase.The glutaraldhyde method conjugating antibody was convenient,efficient and practical.Conclusion:Immunoliposomes with bioactivity of enzyme and antibody are prepared through reasonable methods.