Preparation Of Functionalized Iron-based Nanomaterial And Application In Tumor Diagnosis And Treatment

Nanotechnology has been applied to fighting cancers due to the limitations of traditional cancer treatment methods（surgery,chemotherapy and radiotherapy）,such as local trauma,poor specificity and severe complications.Nanodrug can enhance the permeability of tumor tissue,extend the retention time of drugs and improve effectiveness and safety of anticancer drugs.Nanodrug has been widely used in tumor imaging,diagnosis and treatment,but the nanodrug delivery system exits the problem of uneven distribution and difficult to clear,resulting in nanodrug cannot be effectively used for cancer treatment.Therefore,we are committed to the research and development of functional nanomaterials with high anti-cancer efficacy.Meanwhile,the designed nanomaterials can response to the stimulus of GSH,p H and light to achieve cancer treatment.Moreover,the early diagnosis of tumors is very important for choosing a suitable treatment formula.In view of this,the preparation and development of an integrated diagnosis and treatment nano-platform with excellent performance is of urgent practical significance.In this study,two functional nanomedicines were constructed,and explore its role in tumor treatment.The specific research content is as follows:Part Ⅰ:Preparation of Fe₃O₄-PLGA-Ce6 composite nanoparticles and application in MRI-guided ferroptosis combined with photodynamic therapy for breast cancer treatment.Firstly,ultra-small size Fe₃O₄ nanoparticles with magnetic monodisperse（about 10 nm）were prepared by high temperature thermal decomposition method.The surface of Fe₃O₄ nanoparticles is modified with citric acid to obtain water-soluble Fe₃O₄nanoparticles.Then the photosensitizer chlorin e6（Ce6）and T2 magnetic resonance contrast agent（Fe₃O₄ nanoparticles）were encapsulated in the polylactic acid glycolic acid（PLGA）shell to obtain Fe₃O₄-PLGA-Ce6 nanoparticles.The characterization of Fe₃O₄-PLGA-Ce6 nanoparticles was measured.The successful loading of Ce6 and Fe₃O₄ was verified by ultraviolet-visible spectroscopy,fluorescence emission spectroscopy and X-ray diffraction methods.The results of magnetic resonance（MR）signal showed that the T2 signal of Fe₃O₄-PLGA-Ce6 nanoparticles increases with the increase of iron concentration.The relaxation rate of the nanomedicine reached 105.1s^-1m M^-1,indicating that Fe₃O₄-PLGA-Ce6 nanoparticles can be used as T2 magnetic resonance contrast agent.In addition,Fe₃O₄-PLGA-Ce6 nanoparticles also exhibited p H responsiveness,which indicate the release of the nano drugs in the acidic tumor microenvironment.The results from flow cytometry analysis showed that Fe₃O₄-PLGA-Ce6 can be internalized into tumor cells.Besides,the experimental results of CCK8 detection of cell survival rate,confocal observation of double staining of live and dead cells,and flow cytometry detection of apoptosis effect all showed that Fe₃O₄-PLGA-Ce6 nanoparticles can improve the killing effect of 4T1 cells after laser irradiation.Intracellular mechanism studies have shown that high concentrations of reactive oxygen species（ROS）mediated the death of tumor cells.Fe₃O₄-PLGA-Ce6nanoparticles can dissociate under acidic conditions,release Fe²⁺/Fe³⁺to catalyze intracellular H₂O₂,generate highly toxic hydroxyl radicals（·OH）,and finally induce cell apoptosis.At the same time,the released Ce6 generated a large amount of singlet oxygen（¹O₂）after laser irradiation,which can oxidize with intracellular biological macromolecules and even induced cancer cell death.Subsequently,we investigated whether ferroptosis is involved in the 4T1 cell death by Fe₃O₄-PLGA-Ce6 nanoparticles.After the addition of ferroptosis inhibitor ferrostatin（Fer-1）and deferoxamine（DFO）,the cytotoxicity induced by Fe₃O₄-PLGA-Ce6 nanoparticles in 4T1 was significantly reduced.The datas verified the role of ferroptosis in cancer cell death induced by Fe₃O₄-PLGA-Ce6.The datas of Western blotting and real-time fluorescent quantitative PCR experiments（RT-q PCR）have once again verified the exitance of ferroptosis,which have the charactesitical of manifested in glutathione（GSH）consumption,the decreased expression level of glutathione peroxidase（GPX4）and cystine transporter（SLC7A11）,and the increased expression level of long-chain acyl-Co A synthase 4（ACSL4）.We further explored the ferroptosis/PDT synergistic anti-tumor effect of Fe₃O₄-PLGA-Ce6 nanoparticles in vivo.First,both the 3D tumor cell spheres and living tumor-bearing mouse models showed high solid tumor permeability and retention（EPR effect）,indicating that the nanosystem has good drug delivery behavior in vivo.The results of in vivo experiments showed that laser irradiation can not only trigger photodynamic therapy（PDT）,but also promote ferroptosis.In a conclusion,Fe₃O₄-PLGA-Ce6 nanoparticle is nano-system integrating magnetic resonance imaging/fluorescence imaging/photodynamic therapy/ferrotherapy,and may become a promising nanodrug.Part Ⅱ:Preparation of SAS@PP-Fe³⁺composite nanoparticles and application in MRI-guided ferroptosis combined with photothermal treatment of breast cancer treatment.First,the monomer of dopamine hydrochloride was self-polymerized in alkaline medium to obtain polydopamine nanoparticles（PDA NPs）.PDA NPs and polyethylene glycol（m PEG-NH₂）undergo an addition reaction in alkaline solution to obtain PEGylated PDA NPs（PP）.Then,the ferroptosis inducer sulfasalazine（SAS）and T1 magnetic resonance contrast agent(Fe³⁺)were encapsulated on the PP throughπ-πelectrostatic interaction and chelation to obtain SAS@PP-Fe³⁺nanoparticles.Under the scanning electron microscope,SAS@PP-Fe³⁺nanoparticles showed a spherical-like morphology with uniform size,and the particle size was about 85 nm.In addition,the dynamic light scattering results showed that SAS@PP-Fe³⁺still has good stability after being stored in water for 8 days.Subsequently,the photothermal effect of SAS@PP-Fe³⁺at different concentrations and different power desinities were examined.The SAS@PP-Fe³⁺with a concentration of 200μg/m L can achieve a good photothermal effect at a laser power of 1.5 W/cm² for 10 min,and its photothermal conversion efficiency can reach to 34.4%.After four heating-cooling cycles,SAS@PP-Fe³⁺still maintained a good photothermal conversion capacity,indicating that SAS@PP-Fe³⁺is an excellent photothermal conversion agent.Next,we studied the toxic effect of SAS@PP-Fe³⁺in 4T1 cells.The results of CCK8 survival rate detection experiment,confocal observation of live and dead cell staining and flow cytometry analysis of toxic effects all showed the best anti-cancer effect ofthe SAS@PP-Fe³⁺group with 808 nm laser irradiation can more obviously kill tumor cells.In order to clarify the mechanism of SAS@PP-Fe³ in 4T1 cells,we detected the production of reactive oxygen species（ROS）.The experimental results showed that the cytotoxic effect of SAS@PP-Fe³⁺-induced was mainly mediated by ROS.Subsequently,we investigated whether ferroptosis participated in the 4T1 cell death of SAS@PP-Fe³.In the experiment,the survival rate of cells treated with glutathione（GSH）and cystine（Cys）increased significantly,while the cell death rate after treatment with glutamic acid（Glu）increased.The results of western blotting and RT-q PCR experiments showed obviously up regulated GPX4 and SLC7A11 in 4T1 cells,and inhibited the expression level of ACSL4 in cells.But the addition of glutamate（Glu）appeared the opposite effect,indicating that SAS@PP-Fe³⁺can promote the occurrence of cell ferroptosis by inhibiting the expression of cystine glutamate antiporter.In addition,compared with the cells treated with SAS@PP-Fe³⁺only,the survival rate of the cells after adding deferiprone（DFP,an iron chelator）is also significantly improved,which may be the Fenton reaction was suppressed.We also further explored the effect of SAS@PP-Fe³⁺in killing tumor cells.The experimental results showed that the expression of lipid reactive oxygen species（LPO）and cysteine-containing aspartate proteolytic enzyme 3（caspase3）increased significantly,SAS@PP-Fe³⁺combined with PTT treatment,confirming the synergistic effect of ferroptosis/PTT.Finally,we studied the ferroptosis/PTT synergistic anticancer effect of SAS@PP-Fe³⁺in vivo.The in vivo FL and MR imaging experiments demonstrated that SAS@PP-Fe³⁺has good tumor targeting ability,and guiding the in vivo dual-modality of ferroptosis/PTT to enhance cancer therapy.The prepared SAS@PP-Fe³⁺nanoparticles have excellent performance,acid tumor microenvironment response and light stimulation response,which can provide a reference for the design of nano-platform integrated diagnosis and treatment.