The Surface Coating Of Ceria Nanoparticles For The Application Of Radioprotection And Photodynamic Therapy On Cancer

Backgroud and ObjectivesCerium oxide nanoparticles(CNPs)are the oxide of rare earth elements in the lanthanide group.The existing of either the +3(fully reduced)or +4(fully oxidized)state of cerium atom in CNPs which can mutate quickly and expediently result in their excellent catalytic activities and the multienzyme mimetic properties,and have emerged as a fascinating and lucrative material in biological fields.However,it is well known that nanoparticles without sufficient surface protection inevitably encounter many obstacles in vivo,especially aggregation and clearance by endothelial reticular system(ERS).Therefore,the surface coating of CNPs is essential to improve the nanoparticle stability and biocompatibility.However,most of polymers and functional molecules,especially the polyethylene glycol(PEG)that could improve many of the biological properties of nanoparticles could not be directly grafted onto CNPs because of the low inherently binding affinity.Therefore,it is necessary to find a robust anchor to bridge up the nanoparticle and the PEG chain.A simple and available method of the surface coating of CNPs should be explored.Ionizing radiation exposure to living tissue generates free radicals resulting in cell damages.Recently,CNPs have shown tremendous potential in radioprotection because of their unique regenerative antioxidant activity and multienzyme mimetic properties.However,some obstacles of naked CNPs lead to its low radioprotective capability.Therefore,the radioprotection of PEGylated CNPs should be studied,and a novel and effective radioprotective agent with minor side effects need to be explored.Photodynamic therapy(PDT)is that photosensitizers(PSs),which accumulate in pathognostic tissues,are energized by light of the appropriate wavelength and transfer photon energy to biological substrates to generate reactive oxygen species(ROS),leading to cytotoxicity.As a noninvasive medical technology,PDT exhibits highly localized tissue damage and relatively minimal side effects which can treat numerous diseases and overcome drug-resistant cancers.However,the use of PDT is associated with different problems,because of hydrophobic characteristics and poor tumor targeting of the PSs,hindering the administration and efficiency of the treatment.The application of CNPs as a nanocarrier for the delivery of PSs has great potential in PDT not only by taking advantages of their high surface area per unit volume,selective targeting,and long circulation time,which are provided by most of other nanosized carriers,but also by occupying the excellent catalytic properties,the synergetic antitumor capability,and/or the high ability of oxygen store which can contribute to the excellent phototoxicity of PSs-conjugated CNPs.However,the application of CNPs for PDT was fewer studied.Therefore,a multifunctional PSs delivery system based on CNPs which was developed for PDT to tumor therapy and overcome drug-resistant,the mechanism studies,and exploration of a novel,effective and targeting PSs delivery system with minor side effects in PDT are essential for the design of clinical PDT protocols.Methods1.The CNPs were prepared by microemulsion or thermal decomposition method,and the succinic acid(SA)and PEG surface coating was developed by probing the interaction between alendronate(AL)anchor and nanoparticles.The dispersibility,stability,existence of functional groups,oxidation state,SOD mimetic activity and cytotoxicity of the CNPs before and after surface coating were analyzed.Besides,the blood circulation retention time and organ distribution of nanoparticles in mice were evaluated.2.Normal human liver cells(L-02)were incubated with CNPs or PEGylated CNPs(CNPs-AL-PEG)followed by 60Co-gamma rays irradiation,and the cell viability,apoptosis,ROS production,and the extent of DNA damage of irradiated cells were evaluated.Cells exposed to different concentrations of the nanoparticles,and the SOD2 protein expression,cellular uptake,and cell localisation were analysed.3.Branched polyethylenimine(BPEI,MW=1800)was first conjugated to PEGylated CNPs,and chlorin e6(Ce6)was then conjugated to polyethylenimine-PEGylated CNPs(PPCNPs-Ce6).The stability analysis,XPS,fluorescenceemission spectra,UV-vis spectroscopy analysis,ζ potentials and the 1O2-producing capacities of materials were performed for characterizations.The cellular uptake and cell localisation of PPCNPs-Ce6 in human cervical cancer cells(Hela)were analyzed.Cells were then incubated with series concentrations of PPCNPs-Ce6 followed by or not administration of 660 nm laser irradiation,and the cell viability,apoptosis,ROS production,lysosomal function,and transmission electron microscope(TEM)were performed for the studies of chemocytotoxicity and phototoxicity.For in vivo studies,athymic nude mice with subcutaneous HeLa cells xenografts were intratumorally injected with PPCNPs-Ce6 followed by NIR irradiation,the tumor volume growth,H&E-stain of tumors and major organs sections were performed.4.A multifunctional drug delivery system based on Ce6/folic acid(FA)-loaded branched polyethylenimine-PEGylation ceria nanoparticles(PPCNPs-Ce6/FA)was developed.The stability analysis,XPS,fluorescenceemission spectra,UV-vis spectroscopy analysis,fourier transform infrared spectroscopy(FT-IR),ζ potentials and the 1O2-producing capacities of materials were performed for characterizations.The effect of low-dose PDT based on PPCNPs-Ce6/FA on the cellular uptake of doxorubicin(DOX),the P-glycoprotein(P-gp)expression and cell viability were evaluated.The cellular uptake and cell localisation of PPCNPs-Ce6/FA in MCF-7/ADR cells were analyzed.Cells were incubated with series concentrations of PPCNPs-Ce6/FA followed by the 660 nm NIR irradiation,and the cell viability,apoptosis and ROS production were measured.Then,the active cleaved caspase-3/-7 and LC3-II protein expression were analysed,the integrity of lysosomes were tracked,the live imaging and the Bio-TEM analysis of irradiated cells were performed,and the intracellular Ca2+ and ATP were measured for cell death pathway studies.For in vivo studies,athymic nude mice with subcutaneous MCF-7/ADR cells xenografts were intravenous administrated of PPCNPs-Ce6/FA followed by 660 nm NIR irradiation.Fluorescence imaging of mice or organs was performed and the tumor volume growth was monitored.For biological toxicity analysis,mice were intravenously injected with PPCNPs-Ce6/FA.The H&E staining of major organs,the blood routine assessments and analyses of hepatic and renal function were performed.Results1.The CNPs with 2-5 nm of diameter were prepared by microemulsion or thermal decomposition method.The bisphosphonate group of AL was found to possess high binding affinity to CNPs.Using alendronate as an anchor,SA and PEG could be easily grafted onto CNPs.After surface coating,the agglomerated CNPs were disassembled from each other suggesting improved dispersibility of the nanoparticles in water.Furthermore,the surface coated CNPs exhibited excellent stabilities in various physiological solutions as no obvious size change was observed in one month.The results also indicated that the nanoparticles after surface coating showed higher SOD activity than the naked CNPs which could be ascribed to the increase of Ce3+ concentration in the nanoparticles.As expected,full retention of cell viability was observed for surface coated CNPs,while the naked CNPs exhibited obvious inhibitory effects on liver cells.In vivo studies indicated that the PEGylation not only prolonged the blood circulation retention time of the CNPs but also changed the finial distribution of the nanoparticles in organs.2.CNPs-AL-PEG exhibited excellent dispersibility and stability in various physiological solutions.PEGylated CNPs exhibited higher concentration of Ce3+ ions but less cytotoxic to L-02 cells than naked CNPs.PEGylated CNPs provided higher radioprotection and reacted with O2·-and H2O2 induced by irradiation more efficiently than naked nanoparticles.PEGylation contributed to the elevated expression of SOD2 in L-02 cells,leading to CNPs-AL-PEG against oxidatively generated DNA damage more efficiently than naked CNPs.Furthermore,PEGylation significant reduced the cellular uptake of nanoparticles(~400 folds)and ameliorated intracellular distribution of nanoparticles.CNPs-AL-PEG was mainly diffused in the mitochondria or cytoplasm while naked CNPs extreme aggregated in lysosomes.3.PPCNPs-Ce6 with high PSs loading(Ce6 vs cerium 40 wt %)exhibited a high degree of solubility and stability in aqueous solutions,and significantly enhanced the delivery of Ce6 into cells and its accumulation in lysosomes,remarkably improving PDT efficacy levels compared to those in the administration of free Ce6 at ultralow drug doses(200 nM).Interestingly,PPCNPs-Ce6 efficiently induced HeLa cell death even at low concentrations(10 μM)without the use of laser irradiation and exhibit chemocytotoxicity.ICP-MS and Bio-TEM analyses demonstrated that ceria nanoparticles enter cells abundantly and accumulate in lysosomes or large vesicles.The evaluation of the effects of the different materials on lysosomal integrity and function revealed that PPCNPs-Ce6 catastrophically impaired lysosomal function compared to results with PPCNPs and Ce6.Studies of apoptosis revealed greater induction of apoptosis by PPCNPs-Ce6 treatment.In vivo studies showed PPCNPs-Ce6 exhibited high levels of efficacy in imaging-guided synchronous photochemotherapy at minimal drug doses and with minimal side effects.4.A multifunctional drug delivery system PPCNPs-Ce6/FA with satisfactory biocompatibility was synthesized for enhanced PSs loading and targeted PDT to overcome drug-resistant breast cancer.The nanocarrier delivery and FA targeting promoted cellular uptake of PSs,which selectivity accumulated in lysosomes and could be triggered by a 660 nm laser to produce ROS,leading to decreased expression of P-gp drug-efflux pumps.This phenomenon promoted the chemotherapeutic efficacy of DOX and enhanced phototoxicity in drug-resistant human breast cancer cells.Investigations of the cell death pathway indicated that the induction of apoptotic or autophagic processes by lysosomal-PDT based on PPCNPs-Ce6/FA occurred in a dose-dependent manner,with a transition to an oncotic pattern of cell death at higher doses,which was accompanied by disruption of lysosomal membrane,cellular membrane blebbing,cell and organelle swelling,cytoplasmic vacuole accumulation,and a robust loss of cellular energy.In the end,studies of the efficacy of PDT in MCF-7/ADR tumor-bearing nude mice indicated that sensitization with PPCNPs-Ce6/FA resulted in significant tumor targeting and increased the rate of tumor inhibition with fewer side effects.In addition,the nanotoxicology analysis indicated that the administration of PPCNPs-Ce6/FA at an appropriate concentration was safe for biological applications.The present investigation provided a new strategy for PDT based on PPCNP-Ce6/FA nanocomposites to overcome drugresistant breast cancers and suggested a new cell death pathway in lysosomal-PDT that could help guide the development of clinical treatment protocols.Conclusion1.Using alendronate as an anchor,functional molecules could be easily grafted onto CNPs leading to enhanced enzyme mimetic activity,reduced cytotoxicity,improved stability,prolonged blood circulation,and decreased liver and especially spleen accumulation of the nanoparticles.2.The PEGylated CNPs(CNPs-AL-PEG)exhibited enhanced Ce3+/Ce4+ ratios,improved intracellular dispersion and ameliorated intracellular-distribution of the nanoparticles,which contributed to the elevated expression of SOD2,the increased protection of cells against ROS and the relieved oxidatively generated DNA damage,leading to the enhanced protecting ability of the nanoparticles against radiation-induced damage of L-02 cells compared to that of naked CNPs.3.The multifunctional theranostic platform(PPCNPs-Ce6)significantly enhanced the delivery of Ce6 into cells and its accumulation in lysosomes,remarkably improving PDT efficacy at ultralow drug doses,further more,PPCNPs-Ce6 exhibited chemocytotoxicity even at low concentrations without laser irradiation,leading to effective cancer treatments involving the use of imaging-guided synchronous photochemotherapy.4.FA targeting made further efforts to the promotion of the cellular uptake of PPCNPs-Ce6/FA.Sensitization of PPCNPs-Ce6/FA resulted in the overcoming of the resistance of MCF-7/ADR cells and the excellent phototoxicity even at ultralow doses.Moreover,NIR-triggered lysosomal-PDT based on the higher dose of PPCNPs-Ce6/FA stimulated an oncosis-like cell death pathway,despite the occurrence of apoptotic or autophagic mechanisms at lower drug doses.