Construction Of Novel Loaded ¹⁷⁷Lu-Ce6-Luminol Liposomes And Their Role In Lung Tumor Therapy

As one of the major causes of threats to human life and health,the prevalence and mortality rate of lung malignancies continue to increase year by year.The traditional treatments for lung malignancies are surgery,external radiotherapy and chemotherapy,all of which have made significant progress in recent years,but the overall survival rate of patients is still not high,and there is an urgent need to find new treatment means.The current rise of emerging technologies such as immunotherapy,targeted therapy and photodynamic therapy has brought new hope to the treatment of lung malignancies.As a newly emerged tumor treatment in recent years,photodynamic therapy is to kill tumors by causing tumor cells to produce high levels of reactive oxygen species,which has the unique advantages of few side effects and little or no damage to healthy tissues.However,conventional photodynamic therapy requires extracorporeal laser irradiation,and some patients will have photoallergic reactions,and the poor hydrophilicity of photosensitizers leads to their low delivery efficiency.The rapid development of nanotechnology in recent years has undoubtedly provided effective help to solve the above difficulties.The group has synthesized the in situ photodynamic therapy material Ce6-Luminol（CL）,which has the advantage of activating the photosensitizer at the tumor site for in situ photodynamic therapy without external light source stimulation.Based on this,in this study,liposomes loaded with radionuclide ¹⁷⁷Lu and in situ photodynamic therapy material CL were constructed to achieve simultaneous radionuclide internal irradiation and photodynamic therapy for tumors and to improve the tumor killing effect.Methods1.Synthesis and characterization of in situ photodynamic therapy material Ce6-Luminol（CL）The in situ photodynamic material Ce6-Luminol（CL）was obtained by linking Ce6 with luminol through amide reaction.The material structure was analyzed by FT-IR,¹H-NMR spectroscopy,and MALDI-TOF-MS mass spectrometry.2.Synthesis and characterization of ¹⁷⁷Lu-Ce6-Luminol loaded liposomes(CLL-¹⁷⁷Lu)Photodynamic therapy liposomes（CLL）and liposomes loaded with ¹⁷⁷Lu-Ce6-Luminol(CLL-¹⁷⁷Lu)were synthesized by thin-film hydrophoresis and remote control,and the effect of carrier materials on the encapsulation rate of ¹⁷⁷Lu and CL was investigated by setting different feeding ratios(¹⁷⁷Lu/DPPC,m Ci/mg).The particle size and potential of liposomes were measured at 25℃using a Malvern Zetasizer Nano ZS instrument,and the morphology of liposomes was observed by transmission electron microscopy.The in vitro stability of CLL-¹⁷⁷Lu in different solvents was investigated at the optimal feeding ratio.3.In vitro cellular assay of CLL-¹⁷⁷LuAfter co-incubation of the tumor cell with the drug,the cytophagocytic effect,in vitro stimulated tumor cell ROS generation ability,in vitro stimulation of macrophage conversion capacity to M1 type and in vitro tumor cell killing effect were investigated.4.In vivo biological distribution experiments of CLL-¹⁷⁷LuThe%ID/g（mean±SD）values were calculated by euthanizing mice and collecting tumors and other tissues at different times after injection of ¹⁷⁷Lu-DOTA or CLL-¹⁷⁷Lu into the tail vein of mice modeling lung metastases from breast cancer and lung cancer transplantation tumors.Fluorescence imaging was performed at 24 h and 48 h to determine the in vivo distribution of CLL-¹⁷⁷Lu and equivalent doses of CL after injection into the model mice through the tail vein,respectively.5.Therapeutic experiments of CLL-¹⁷⁷Lu on mice with transplanted tumor model of lung cancer and mice with lung metastasis model of breast cancerThe model mice were divided into control,low-dose,medium-dose and high-dose groups.Different doses of CLL-¹⁷⁷Lu were administered to each group of mice through the tail vein,and the injection was repeated once after 7 days.The mice were euthanized 5 days after the second administration,and the tumor treatment effect was evaluated as well as the tumor site ROS content,M1 type macrophage percentage and tumor tissue apoptosis were measured.The final treatment dose of the model mice was selected by the above experiments to treat the mice.The mice were divided into control group,¹⁷⁷Lu group,CLL group and CLL-¹⁷⁷Lu group,and the above steps were repeated.6.In vivo safety evaluationThe in vivo safety of CLL-¹⁷⁷Lu was evaluated by body weight,organ indices,blood cell counts,serum enzymology and pathology of major tissues in mice.Results1.FT-IR,¹H-NMR spectroscopy,and MALDI-TOF-MS mass spectrometry all confirmed that the in situ photodynamic material Ce6-Luminol was successfully synthesized（CL）.2.CLL-¹⁷⁷Lu was successfully prepared and characterized in terms of particle size and potential at four feeding ratios of 5:10,10:10,20:10,and 40:10（m Ci/mg）.The best feeding ratio was determined to be 20:10,and the target material CLL-¹⁷⁷Lu had a lipid bilayer structure,particle size of about 140 nm,and a negatively charged surface.The particle size of CLL-¹⁷⁷Lu remained basically stable when co-incubated with different solvents for 72 h,showing good in vitro stability.3.By comparing the cell survival rate of tumor cells after co-incubation with ¹⁷⁷Lu,CLL or CLL-¹⁷⁷Lu,it was shown that the killing effect of CLL-¹⁷⁷Lu after 72 h co-incubation with4T1 cells or A549 cells was enhanced 2-3 times compared with ¹⁷⁷Lu group or CLL group alone（26%survival of 4T1 cells and 17%survival of A549 cells at this time）,and the killing effect was dose-dependent.4.In vitro flow cytometry and fluorescence confocal results showed that the phagocytosis of tumor cells was significantly enhanced by liposome-encapsulated drugs,and for unencapsulated ¹⁷⁷Lu,the phagocytosis of tumor cells was increased approximately 11-12 times after encapsulation using liposomes,and the phagocytosis was time-dependent;relative to ¹⁷⁷Lu and CLL alone,the ability of CLL-¹⁷⁷Lu to stimulate tumor cells to produce ROS was increased about 2-5 times and the ability to stimulate macrophage conversion to M1 type was increased about 1.2 times,and this ability was dose dependent.5.The%ID/g values of isolated tissues of mice with lung metastasis model of breast cancer and mice with transplantation tumor model of lung cancer were calculated,which showed that the use of liposomes to encapsulate and ¹⁷⁷Lu significantly prolonged their circulation time and increased their aggregation in tumor sites,the amount of nucleophiles aggregated at the tumor site after 48 h of administration was about 20-50 times that of uncoated nucleophiles.The fluorescence imaging results of the model mice showed that liposome encapsulated CL significantly increased its aggregation at the tumor site,and its aggregation at the tumor site after 48 h of administration was about 2.5 times that of unencapsulated CL.6.High,medium and low dose of CLL-¹⁷⁷Lu had a certain degree of therapeutic effect on lung cancer transplantation tumor model mice and breast cancer lung metastasis model mice.Compared with the CLL,¹⁷⁷Lu group alone,the CLL-¹⁷⁷Lu group had approximately 1.5 times higher ability to stimulate ROS production at tumor sites,2.5 times higher ability to stimulate the conversion of macrophages to M1 type at tumor sites,and 1.5 times higher ability to induce apoptosis in tumor tissues,and the above abilities were CLL-¹⁷⁷Lu concentration-dependent.7.In vivo experiments verified that the nanomedicine CLL-¹⁷⁷Lu mice showed no significant changes in body weight,organ index,blood cell count,serum enzymology and pathological examination of major tissues,with good in vivo biosafety.Conclusions1.The in situ photodynamic material Ce6-Luminol（CL）was successfully prepared.The liposomes were used to synthesize the target liposome CLL-¹⁷⁷Lu by successfully accomplishing the encapsulation of the target nucleophile ¹⁷⁷Lu and the intercalation of CL at the same time,and it has a good in vitro stability.2.In vitro experiments verified that CLL-¹⁷⁷Lu can increase phagocytosis of tumor cells,can stimulate tumor cells to produce high levels of ROS,and can increase more conversion of macrophages to M1 type,which has a better killing effect on tumor cells.3.CLL-¹⁷⁷Lu has a longer circulating half-life and accumulates more at the tumor site in mice,and the tumor model mice demonstrated that CLL-¹⁷⁷Lu can stimulate tumor tissue to produce high levels of ROS,stimulate tumor-associated macrophages to convert to M1 type,and have a better killing effect on tumor tissues.4.Preliminary safety tests show that CLL-¹⁷⁷Lu has a good biosafety.In summary,a liposome CLL-¹⁷⁷Lu loaded with ¹⁷⁷Lu and Ce6-Luminol was constructed,and its combined therapeutic effect on tumors with radionuclide internal irradiation therapy and photodynamic therapy was confirmed by in vitro and in vivo experiments.This nanomaterial is easy to prepare and has good biosafety,which is expected to be a new tool for the treatment of malignant tumors.