Long-circulating Liposomes Loaded With ?-elemene And Photosensitizers For Phototherapy-chemotherapy Synergistic Antitumor Studies

Malignant tumors are a serious threat to human health,and chemotherapy is one of the main treatment methods in clinic.The commonly used chemo-agents can inhibit the proliferation of cancer cells or effectively kill them by interfering with the anabolism of nucleic acid,suppressing cell mitosis and protein synthesis.However,there are also increasing literatures reporting the limitations of chemotherapy,such as low selectivity,adverse side effects on normal tissues,and emergence of drug resistance which have greatly compromised the therapeutic outcome.Therefore,to find and develop safer and more effective chemo-agents is of great importance in clinic,?-elemene(ELE),a sesquiterpene compound extracted from the traditional herbal medicine Curcumae Rhizoma,is an important plant-derived anti-tumor drug.ELE has shown suppressive effects on a variety of tumors,and has minimal side effects.In clinical application,ELE was used alone or combined with other chemo-agents to treat various tumors,including lung cancer,gastrointestinal cancer,breast cancer and etc.To further improve the efficacy of cancer treatment,combination therapy has become an attractive strategy in recent years,such as chemotherapy combined with surgery,radiotherapy,phototherapy and immunotherapy.Phototherapy is a tumor treatment method characterized by non-invasiveness and high selectivity.And it includes photothermal therapy(PTT)and photodynamic therapy(PDT).The principle is to activate the photosensitizers accumulated in tumor tissues by the near infrared(NIR light)at a specific wavelength.PTT could effectively ablate tumors via the heat generated by photosensitizers upon NIR light irradiation.And PDT relies on the ROS generated by photosensitizers under NIR light irradiation to induce tumor cell apoptosis and necrosis.In this study,we hypothesized that by combining the chemotherapy mediated by ELE and phototherapy achieved by IR780,a significantly enhanced anti-tumor efficacy could be obtained.IR780 is a small molecular near-infrared photosensitizer which exhibits excellent photothermal and photodynamic effects.It has attracted increasing attention in the field of tumor imaging and treatment.However,due to its lipophilic nature,free IR780 is extremely difficult to be dissolved in water and this severely limits its further application in clinic.And the toxicity of free IR780 is also of great concern due to its low maximum tolerated dose in the present reports.In addition,the clinical application of ELE has also been limited by its poor solubility in water,low bioavailability and strong irritation to blood vessels.Therefore,it is challenging to co-deliver the IR780 and ELE to the tumor tissue efficiently for combined chemo-phototherapy.In recent years,long-circulating liposomes have shown great prospects in the field of drug delivery due to their tumor-targeting ability,long systemic circulation and good biocompatibility.In this study,IR780 and ELE were co-loaded into the long-circulating liposomes to prolong their circulation time,increase tumor accumulation and achieve combined chemo-phototherapy.This thesis includes three parts and the specific content of each part was listed below.1.Preparation and characterization of IR780-ELE-LCLFirstly,the analytical methods of ELE and IR780 were established by HPLC and visible spectrophotometer,respectively.And methodological investigations of linear relationship,precision,repeatability,stability and sample recovery were carried out.Results showed that the established methods were suitable for the determination of ELE and IR780.The longcirculating liposomes co-loaded with IR780 and ELE(IR780-ELE-LCL)were prepared by the thin film hydration method,and then the characterization of IR780-ELE-LCL was carried out.The morphology of IR780-ELE-LCL was observed by a transmission electron microscope,and the results showed that IR780-ELE-LCL was uniformly-sized spheres with a smooth surface and good dispersibility.The particle size and zeta potential of the samples were measured by Zeta Sizer Nano-ZS90,and the results showed that the particle size of IR780-ELE-LCL was 130.03 nm ±1.55 nm with PDI of 0.223 ± 0.011,and the zeta potential was-1.48 mV ± 0.26 mV under pH 7.4.The encapsulation efficiency and drug loading of IR780-ELE-LCL were measured by filter retention method and ultracentrifugation method.The results showed that the encapsulation efficiencies of ELE and IR780 in IR780-ELE-LCL were 90.23%± 0.80%and 85.11%± 0.34%,respectively,and the loading efficiencies were 9.75%±0.08%and 0.47%± 0.01%,respectively.The physical stability of IR780-ELE-LCL was investigated through measuring the change in particle size.It was found that when being stored under 4? away from light,the particle size of IR780-ELE-LCL did not change significantly within 7 days,indicating relatively good physical stability.Subsequently,the photo-stability of IR780 in IR780-ELE-LCL under different storage conditions was investigated by a visible spectrophotometer,and the results showed that IR780-ELE-LCL could remain stable within 7 days at 4? under dark conditions.To study the in vitro photothermal effect,samples were exposed to laser irradiation at 808 nm(2W/cm2),and the temperature change was recorded by an IR camera and analyzed using the FLIR Tools software.The results demonstrated that IR780-ELE-LCL had an excellent photothermal conversion efficiency,and the magnitude of changing in temperature was positively correlated with the concentration of IR780,laser irradiation time and the power density of laser.The SOSG fluorescent probe was used to detect the yield of singlet oxygen.The results showed that IR780-ELE-LCL could produce a large amount of singlet oxygen under the 808 nm laser irradiation,indicating that it has a strong photodynamic effect.2.Evaluation of the cytotoxic effect of IR780-ELE-LCL in vitroThe cytotoxicity of ELE-LCL against different tumor cells was investigated by MTT assay.Among them,?-elemene had a stronger inhibitory effect on the lung cancer cells,and the IC50 value of ELE-LCL for the A549 cells and LLC cells were 35.17?g/mL and 14.17 ?g/mL,respectively.Due to the low tumor formation rate of A549 cells,LLC cells were used as the cell model to study the anti-tumor effect of IR780-ELE-LCL in the following experiments.The MTT assay was also used to compare the cytotoxicity of chemotherapy,phototherapy and the combined chemo-phototherapy against LLC cells.Under the same ELE concentration,IR780ELE-LCL plus laser irradiation showed much stronger cytotoxicity compared to single chemotherapy(ELE-LCL)or single phototherapy(IR780-LCL plus Laser).This result confirmed the enhanced anti-tumor effect of IR780-ELE-LCL for combined chemophototherapy.The live and dead assay was used to further confirm the results of the cytotoxicity experiment.Cells became all dead after the IR780-ELE-LCL plus laser treatment,which demonstrated that the combined chemo-phototherapy could kill the cancer cells more effectively.The reactive oxygen species fluorescent probe DCFH-DA was used to detect the level of reactive oxygen species in the cells.The results showed that IR780-ELE-LCL could produce a large amount of reactive oxygen species in the cells under the 808 nm laser irradiation,and its yield of active oxygen species was higher than other treatment groups.3.Evaluation of the anti-tumor efficacy of IR780-ELE-LCL in vivoTo study the in vivo distribution of IR780-ELE-LCL and its anti-tumor efficacy,a subcutaneous LLC tumor model was established using C57BL/6J mice.Free IR780 and IR780ELE-LCL with IR780 dosage of 2 mg/kg were intravenously injected into the mice,and the fluorescence signal was recorded by the small animal live imaging system.Compared to free IR780 group,the fluorescence signal of IR780-ELE-LCL group in tumor tissues was significantly enhanced,indicating that the long-circulating liposomes could improve the accumulation of drugs and photosensitizers within tumor tissues via the enhanced permeability and retention effect(EPR effect).At 12 h post-injection,the tumor area was exposed to laser irradiation(808 nm,2W/cm2),and the temperature change of tumor area was recorded by an IR camera.For the IR780-ELE-LCL treated mice,the temperature of tumor area reached up to 54?,which could effectively ablate the tumor cells.This confirmed that IR780-ELE-LCL could exhibit photothermal effect in vivo.Then,the anti-tumor efficacy was evaluated on the LLC xenograft model.Compared to control group,the IR780-ELE-LCL treated group did not show obvious inhibition on the tumor growth,and this was probably because that the amount of ELE within the tumor tissues was not insufficient to kill the tumor cells effectively.For the IR780-LCL+Laser treated group,obvious tumor suppression was observed due to the hyperthermia induced by IR780.The IR780-ELE-LCL+Laser treated group showed the most effective tumor growth inhibition compared to single chemotherapy or phototherapy.This result confirmed the enhanced anti-tumor efficacy of combined chemo-phototherapy mediated by IR780-ELE-LCL.The tumor weight measurement showed that the tumor inhibition rates(TIR)of IR780-ELE-LCL,IR780-LCL+Laser and IR780-ELE-LCL+Laser treated group were 19.92%,73.37%and 85.82%,respectively.Finally,the main organs were collected and sliced for observation after H&E staining.The results showed that there were no obvious pathological changes in the heart,liver and kidney of mice for each treatment group,indicating that administration of IR780-ELE-LCL was relatively safe.In summary,the long-circulating liposomes co-loaded with the photosensitizer IR780 and the chemo-agent ELE were prepared by the thin film hydration method,and the anti-tumor effect of IR780-ELE-LCL for combined chemo-phototherapy was investigated by cell experiments and animal experiments.In this study,the IR780-ELE-LCL plus laser irradiation exhibited the strongest cytotoxicity in vitro as well as the significantly enhanced anti-tumor efficacy in vivo.IR780-ELE-LCL could achieve co-delivery of chemo-agents and photosensitizers to the tumor sites,providing experimental and reference basis for combined chemo-phototherapy to combat cancer in clinic.