A Manganese Nanoparticle,PEG-Mn-BDA,Selectively Inhibited Cancer Via Mitochondrial Mechanisms And Induced Fluorescence And Magnetic Resonance Imaging

Background: Cancer is a leading cause of death in the world.The burden is expected to grow worldwide due to the growth and aging of the population,particularly in less developed countries,in which about 82% of the world's population resides.Although there has been the development of a plethora of chemotherapeutic agents,we still need to develop more.The limitations of current anticancer drugs are due to the relative ineffectiveness of the treatment as well as side effects,increased incidence,and rapid development of drug resistance.Coupled with these problems are late diagnosis of cancer and difficulty in confirming margin in cancer surgery.Aim: The aim of this study is(I)to synthesize and characterize a manganese polyethylene glycol nanoparticle,labeled as(PEG-Mn-BDA);(II)to detect the anticancer effect and side effect of PEG-Mn-BDA;(III)to analyze mitochondrial mechanisms of PEG-Mn-BDA underlying its selective anticancer effects;(IV)to determine whether PEG-Mn-BDA can induce both fluorescence and MRI imaging in cancer cells.This,when further developed,could be used in both cancer therapy and diagnosis.Methods: I.The PEG-Mn-BDA was synthesized by reacting PEG-NHCH2 COOH with Mn Cl2.4H2 O and m-BDA.The morphology of the nanoparticle was examined by Transmission Electronic Microscope(TEM),the size was measured by dynamic light scattering(DLS),the structure was confirmed by IR and UV spectrophotometers and the content of manganese in PEG-Mn-BDA was measured using atomic absorption spectrometer.II.Different cancer cells lines and normal cell lines were used to assess the inhibitory effects on cell viability by MTT assay and the cell morphology were taken with an inverted microscope.The apoptosis was detected by Annexin-V/PI staining using Flow cytometry,and the autophagy was analyzed by Monodansylcadaverine(MDC)staining under fluorescent microscope.The presence of cell death,proliferation and transportation related proteins were detected by Western blot analysis.Mitochondria function was assessed by JC-1 and Rhodamine 123 dyes for mitochondrial membrane potential(MMP),DCFH-DA for reactive oxygen species(ROS),and luciferin-luciferase assay kit for intracellular ATP level.Glycolysis was analyzed by detection of LDH-A activity using the LDH assay kit.III.The fluorescence images were observed using a Laser Scanning Confocal Microscope while MRI images were taken using Trio 3.0 TMR Imager.IV.A mouse xenograft model was established by transplanting Hep-A cells in ICR mice.Preliminary anticancer effects and side effects were assessed.Main results were shown as follows: 1.Adpa-Mn could effectively and selectively exert anti-proliferative effect on glioma cells in both dose and time dependent manner which was better than that of cisplatin.The selectivity was found to be dependent on Tf R-Tf system which was highly expressed on glioma cells.Adpa-Mn was able to interact with mitochondria by depolarizing mitochondrial membrane potential and reducing ATP production.Adpa-Mn induced apoptosis as pro-apoptotic proteins,caspase 3,caspase 7 and caspase 9 were highly expressed while anti-apoptotic protein,Bcl2 expression was greatly reduced and flow cytometry analysis revealed increasing apoptotic cell death.But autophagy induced by generation of ROS was not involved in its anticancer mechanisms.2.m-BDA could effectively and selectively exert anti-proliferative effect on SMMC-7721,HCT-116 and Hep G2 cells in both dose and time dependent manner.m-BDA was absorbed into the cytosol but not the nucleus of the cell and showed fluorescent imaging.m-BDA was able to interact with mitochondria by depolarizing mitochondrial membrane potential which contributed to the apoptotic cell death as caspase 3 expression increased.m-BDA was also able to inhibit LDH-A as the expression and the activity of LDH-A were decreased.The inhibition of LDH-A diverted the pyruvate into the mitochondria which aided in increased ROS generation and induced apoptosis.m-BDA exhibited higher anti-proliferative activity on cancer cells in hypoxic conditions than in normoxic conditions.3.PEG-Mn-BDA was successfully synthesized and characterized.Results from the TEM and DLS revealed that the size of PEG-Mn-BDA was about 200 nm.PEG-Mn-BDA kept the inhibitory properties of Adpa-Mn and m-BDA as it could effectively and selectively exert anti-proliferative effect on SMMC-7721,HCT-116 and Hep G2 cells in both dose and time dependent manner.The inhibitory effect of PEG-Mn-BDA was found to be higher than that of m-BDA.PEG-Mn-BDA was absorbed into the cytosol but just as m-BDA but lost part of its fluorescent imaging ability as the intensity was observed to be weaker than that of m-BDA.PEG-Mn-BDA also induced MRI imaging in cancer cell which shows that it could be used for bimodal imaging.Furthermore,PEG-Mn-BDA was able to inhibit LDH-A and decreased its expression and activity.In addition,PEG-Mn-BDA generated intracellular ROS which contributed to the induction of apoptosis as the pro-apoptotic protein,caspase 3 was highly expressed.Moreover,PEG-Mn-BDA was able to depolarize mitochondrial membrane potential just as Adpa-Mn and m-BDA,which also contributed to the apoptotic cell death.PEG-Mn-BDA was found to be more effective on cancer cells in hypoxic conditions than in normoxic conditions.In vivo,PEG-MnBDA could also exert inhibition on a mouse hepatocellular carcinoma xenograft.This makes PEG-Mn-BDA a good candidate for cancer treatment since side effect is minimal.Conclusion Our study clearly indicated that the synthesized PEG-Mn-BDA was found to be effective to treat cancer with minimum side effect through mitochondria.Our results illustrated that PEG-MnBDA could be used for bimodal imaging techniques and was capable of providing solutions to address multiple issues of sensitivity,resolution,and tissue penetration in tumor diagnosis and therapy.Moreover,our study demonstrated that PEG-Mn-BDA was able to target the metabolic pathway of cancer and redistributed the energy via oxidative phosphorylation and therefore has the potential to reduce the occurrence of multidrug resistance.This makes PEG-Mn-BDA a very good candidate for bimodal imaging and therapy of cancer.