Inhalable Mineralized Liposomes Co-loaded With Dihydroartemisinin And Calcium Phosphate For The Treatment Of Lung Cancer

Background:The incidence and mortality of cancer are increasing rapidly worldwide.Among them,the mortality rate of lung cancer with a high incidence ranks first in cancer-telated mortality and it become the leading cause of cancer-related death,posing a serious threat to the health and life of human and deserving our close attention.Chemotherapy is the first choice for lung cancer treatment in clinic.Chemotherapeutic drugs mainly kill tumor cells by inducing apoptosis through the oral administration or intravenous injection,leading to severe side effects and multiple drug resistance.In the past decade,ferroptosis has received extensive attention from researchers as a novel non-apoptotic tumor therapy.Ferroptosis induces cell death through cuasing cellular oxidative stress and the massive accumulation of intracellular iron-dependent lipid peroxides,which could bypass the traditional apoptotic pathway and thereby avoiding the generation of drug resistance.It has been proved that tumor cells are highly sensitive to ferroptosis.Therefore,the development of ferroptosis-based novel lung cancer treatment strategies possess broad application prospects.Fenton reaction or Fenton-like reaction is one of the most widely applied ferroptosis-inducing mechanisms to induce the large amount production of reactive oxygen species(ROS)in the tumor site.Nervetheless,in the ferroptosis therapy for lung cancer based on Fenton reaction,there are three major bottlenecks: the low accumulation of drugs in the lung cancer lesions,the poor drugability of traditional Fenton reaction substrates and the insufficient generation of exogenous ROS by Fenton reaction.Aiming to solve the above three bottleneck problems,an inhalable biomineralized liposome(LDM)was constructed in this study for the endoplasmic reticulum-induced lung cancer ferroptosis therapy.Compared with oral administration and intravenous injection,pulmonary inhalation administration could increase the drug accumulation in the lung cancer lesions by its unique lung targeting ability and reduced systemic toxicity,becoming the most effective administration methods for the lung cancer ferroptosis therapy.Dihydroartemisinin(DHA)-loaded liposomes,a safe and efficient Fenton reaction substrate,could induce the occurrence of ferroptosis by Fenton-like reactions-mediated increase of intracellular ROS levels and the subsequently accumulation of lipid peroxides.The Ca2+ release from calcium phosphate(Ca3(PO4)2,Ca P)mineralized layer coated on the surface of liposomes can trigger the endoplasmic reticulum stress(ER stress)response induced by Ca2+-overload,which could lead to the dysfunction of mitochondria and enhance the generation efficiency of endogenous ROS,leading to the accumulation of lipid peroxide(LPO)of cells and achieving the ER stress-enhanced ferroptosis process.Methods:LDM was prepared with the optimal formulation through liposomes formulation screening and process optimization.The particle size,Zeta potential,encapsulation efficiency,pH-responsive degradability and stability of LDM were determined.In order to investigate the feasibility of LDM as a pulmonary drug delivery system,the immunodeficiency mice were employed to construct an orthotopic lung cancer model based on human A549-luc lung cancer cells transfected with luciferase gene,and the nebulization performance,in vivo distribution and the co-localization of LDM with cancer cells were evaluated by a self-made nebulizer device.The human A549 lung cancer cells were used as the in vitro pharmacodynamic evaluation model of LDM.The cellular uptake and lysosomal escape behavior of liposomes were studied by the confocal laser scanning microscopy and flow cytometry.Besides,the cytotoxic effect of LDM was also studied.In order to investigate the synergistic mechanism of Ca2+ overload-enhanced endoplasmic reticulum stress on ferroptosis,the changes of intracellular Ca2+ content,the expression levels of sarcoplasmic/endoplasmic reticulum Ca2+-ATPases(SERCA)and glucose regulated protein 78(GRP78),Ca2+ overload mitochondria,the activity of mitochondria and ATP levels were determined to explore the Ca2+ overload-enhanced endoplasmic reticulum stress process.The cell death forms oinduced by LDM was comfirmed by the cell death pathway assay.The intracellular ROS levels were determined by confocal laser scanning microscopy and flow cytometry,and the levels of intracellular glutathione(GSH),lipid peroxides and malondialdehyde(MDA)were measured to explore the synergistic effect of endoplasmic reticulum stress on ferroptosis.The size and morphology of cell mitochondria were measured by biological transmission electron microscopy.The integrity of cell membranes was analyzed by various analysis methods.Besides,the surface morphology of the cells was studoed by biological transmission electron microscopy and atomic force microscopy,and the degree of cell swelling and the roughness of the cell surface were also analyzed to further clarified the mechanism of ferroptosis.Finally,an A549-luc orthotopic lung cancer model was constructed based on immunodeficiency mice to systematically evaluated the in vivo efficacy and safety of LDM.Results:The results showed that the prepared biomineralized liposomes LDM were about 150 nm in particle size and possess good distribution uniformity.The encapsulation efficiency of LDM for DHA was close to 100%,which proved an excellent drug-loading capacity.The pH-responsive degradation experiments illustrated that the Ca P in the outer layer of LDM had a satisfactory pH-responsive degradation behavior.The feasibility study of LDM as a pulmonary drug delivery system showed that LDM possessed a remarkable inhalation performance and high deposition behavior in the lungs,demonstrating that LDM could be used as a pulmonary drug delivery system for the treatment of lung cancer.The cellular uptake assay and lysosomal escape behaviors indicated that LDM could be effectively taken up by A549 cells and could successfully escape from lysosome capture through the proton sponge effect mediated by Ca P.Cell viability assays showed that the survival rate of lung cancer cell line A549 was only about 15.82% after LDM treatment for 24 h,illustrating that LDM had efficient ability for tumor cell killing.The study on the mechanism of Ca2+ overload-enhanced endoplasmic reticulum stress elaborated that LDM could induce the occurrence of endoplasmic reticulum stress and the up-regulate the expression level of intracellular GRP78 by Ca2+ overload-induced disturbance of intracellular Ca2+ homeostasis and the blockade of SERCA by DHA.The occurrence of endoplasmic reticulum stress could disrupt the mitochondrial membrane potential and damage the mitochondrial activity through mitochondrial Ca2+ overload,which would ultimately lead to the mitochondrial dysfunction and the down-regulation of intracellular adenosine triphosphate(ATP)levels.In the study of the synergistic mechanism of ferroptosis,the results of cell death pathway analysis demonstrated that the ferroptosis process mediated by oxidative stress played a significant role,and cell apoptosis process was partially involved in.The ROS generation experiment revealed that the intracellular ROS content in A549 cells was increased by 5.22 times after LDM treatment for 12 h,and the intracelullar GSH levels also could be down-regulate to43.95% of the control group.In addition,the levels of key markers in the process of ferroptosis such as LPO and malondialdehyde(MDA)were studied,and the resuls showed that LDM could significantly induce the accumulation of intracellular LPO and increase the MDA content to1.95% of the control group.LDM could significantly induce the accumulation of intracellular lipid peroxides and increase the content of malondialdehyde to 1.95 times that of the control group,successfully achieving the synergistic effect of endoplasmic reticulum stress on ferroptosis.The synergistic mechanism of ferroptosis also elaborated the fact that the mitochondria of A549 cells treated with LDM had classic ferroptosis features such as volume shrinkage,increased mitochondrial membrane density and reduced mitochondrial cristae.The osmotic disturbance induced by Ca2+ overload and the accumulation of lipid peroxides together led to the fragmentation of A549 cell membranes,the swelling of cells and the roughness of cell surface.In vivo anti-tumor efficacy experiments demostrated that LDM could effectively inhibit the growth of tumors in mice through pulmonary inhalation administration,and its tumor inhibition rate was increased up to 79.3%.Besides,LDM couuld remarkablely increase the Ca2+ content in lung cancer lesions,which confirmed that the auxiliary role of Ca2+ overload in the process of enhanced ferroptosis.The tumor pathological analysis studies illustrated that LDM could induce the of apoptosis tumor cell,and it also could inhibit the cell viability,proliferation and invasiveness of tumor cells.The results of in vivo safety analysis showed that LDM through pulmonary inhalation has negligible effect on the body weight,main organs,trachea and blood system of tumor-bearing mice,which confirmed that the excellent biological safety of LDM.Conclusion:In summary,this study develpoed an inhalable biomineralized liposome to achieve an efficient treatment of lung cancer.Upon deposited into the lung cancer lesions through pulmonary inhalation administration,LDM can successfully escape from the lysosomes through the proton sponge effect in the acidic lysosomal environment mediated by the Ca P in the outer layer,releasing a large amount of Ca2+ and loaded DHA.DHA can induce the accumulation of intracellular lipid peroxides and subsequent ferroptosis of tumor cells by increasing the celullar ROS content mediated by Fenton-like reaction.The released large amount of Ca2+ can lead to endoplasmic reticulum stress in tumor cells through Ca2+ overload,and subsequently result in the mitochondrial dysfunction through the imbalance of mitochondrial Ca2+ homeostasis,significantly improving the efficiency of intracellular ROS generation,which was beneficial to the accumulation of LPO and the synergistic activation of ferroptosis in tumor cells.This inhalable biomineralized liposomes not only provides new insights into the treatment of lung cancer through pulmonary inhalation administration of liposomes,but also provides new strategies and new ideas for the efficient treatment of lung cancer in combination with endoplasmic reticulum stress and ferroptosis.