The Construction Of A Novel Targeted Nano Drug-loading System For High-risk Myelodysplastic Syndrome And Its In Vitro Mechanism

OBJECTIVEThe myelodysplastic syndromes(MDS)are a very heterogeneous group of myeloid disorders characterized by peripheral blood cytopenias and increased risk of transformation to acute myeloid leukemia(AML).When patient with MDS transformed to AML(t AML),they had lower complete remission rates and poor survival.There are not targeted drugs for patients with MDS at present.Currently,some studies have found that CD123 antigen was expressed on the surface of leukemia stem cells(LSCs)was considered the new targeted antigen for patients with leukemia.CD123 antigen was highly expressed on the surface of high risk MDS cells.Therefore,CD123 antigen as targeted antigen,CdTe was used as drug carriers and conjugated anti-CD123 monoclonal antibody(antiCD123),as a targeting vector(antiCD123-CdTe).Then antiCD123-CdTe co-loaded with daunorubicin(DNR)(antiCD123-CdTe-DNR)can precisely target high risk MDS cells and deliver DNR.By observing the therapeutic effects of targeting drug delivery system on MUTZ-1 cells,it provides a new theoretical and experimental basis for MDS patients with targeted therapy.METHODSCdTe conjugated antiCD123 through amide bond,co-loaded with DNR with electrostatic bonding.According to encapsulation efficiency and drug loading optimize processing conditions for preparation of antiCD123-CdTe-DNR.Dynamic light scattering(DLS)and Zeta potential were performed to evaluate the changes of particle size and potential before and after drug carrier;the DNR release behavior from CdTe in PBS buffer at different pH conditions.AntiCD123 connection rate of CdTe can be detected by commassie brilliant blue.Confocal fluorescence microscopy was used to observe the effect of targeted system to MUTZ-1 cells and nuclear morphological changes of MUTZ-1 cells.MUTZ-1 cells were co-cultured with control group(PBS),antiCD123 mAb,CdTe,DNR,CdTe-DNR and antiCD123-CdTe-DNR,respectively.Then,flow cytometry was used to detect the average fluorescence intensity and apoptosis of MUTZ-1 cells.The expression of apoptosis-related proteins of MUTZ-1 cells(P53,Cleaved-caspase9,Bcl-2 and Cleaved-caspase3)in each treatment groups were detected by western blotting.RESULTS(1)When the concentration of DNR was 200ug/ml,the drug encapsulation efficiency and drug loading rates reached the maximum(81.72 ± 0.57%;44.97± 0.17).The release rate of DNR from CdTe in PBS buffer at different pH,The most rapid release of DNR was obtained at pH=6.0,and approximately 90% of the loaded DNR was released into the buffer within 24 hours,however,the release was slower at pH=7.4,and approximately 40% of the loaded DNR within 24 hours.(2)AntiCD123mAb could be connected with CdTe and connection rate reached about 70%.When 5ul(100ug/ml)antiCD123m Ab add to the above drug loading system,the drug encapsulation efficiency and drug loading rates reached the maximum(74.52±1.81%;42.08±0.64%).(3)The particle size / Zeta potential of drug carrier(CdTe)and CdTe conjugated antiCD123mAb co-loaded with DNR(CdTe-DNR,anti CD123-CdTe-DNR) were 7.13 ± 1.099 nm /-12.5 ± 1.57 mV,109.11 ± 7.316 nm /-26.23 ± 0.9 mV;114.91 ± 9.749 nm /-32.93 ± 1.0 mV,respectively.(4)MUTZ-1 cells were treated with PBS(control group)、DNR、CdTe-DNR、antiCD123-CdTe-DNR for 4 hours,respectively.AntiCD123-CdTe-DNR could target bind to MUTZ-1 cells was detected by confocal microscope.The average fluorescence intensity of DNR 、 CdTe-DNR and antiCD123-CdTe-DNR increased gradually were detected by flow cytometry,and the differences were statistically significant(P<0.05).(5)MUTZ-1 cells were co-cultured with PBS(control),CD123,CdTe,DNR,CdTe-DNR,antiCD123-CdTe-DNR for 24 hours,respectively.The inhibition of apoptosis of each groups were performed by flow cytometry: 5.06±1.34%、5.56±1.06%、6.3±0.86% in the control group,CD123 group and CdTe group,respectively(P> 0.05),however,the apoptosis rate of DNR group was 30.4 ±3.24%,which was significantly different from that in the above three groups(**P < 0.01).In addition,the apoptosis rate of CdTe-DNR group was 62.06 ±5.23%,there was significant difference compared with DNR group(** P <0.01).The apoptotic rate of antiCD123-CdTe-DNR group had higher than CdTe-DNR group,reached 76.9 ± 3.86%(* P <0.05).(6)MUTZ-1 cells were co-cultured with PBS(control),CD123,CdTe,DNR,CdTe-DNR,antiCD123-CdTe-DNR for 24 hours,respectively.The apoptosis related proteins of P53,Cleaved-caspase9,Bax and Cleaved-caspase3 were detected by Western Blotting.We found upregulation of apoptosis related proteins in the trial groups(DNR,CdTe-DNR,anti CD123-CdTe-DNR)and the highest expression of apoptosis related proteins in the targeted drug-loaded system group(P <0.01).CONCLUSIONIn this study,we constructed an anti-CD123-CdTe-DNR targeting carrier system successfully with high drug loading efficiency and entrapment efficiency.AntiCD123-CdTe-DNR can proactively target CD123 antigen on the surface of MUTZ-1 cells,which can effectively transport DNR into MUTZ-1 cells.AntiCD123-CdTe-DNR can significantly enhance the apoptosis rate of MUTZ-1 cells induced by DNR through up-regulating the apoptosis protein levels of P53,Cleaved-caspase9,Bax and Cleaved-caspase3.