Study On Applications In Cancer Photodynamic Therapy Of Gd₂O₃ Upconversion Fluorescent Nanomaterials

Photodynamic therapy?PDT?has become a research hotspot due to its advantages including better targeting,less-injury,higher efficiency,better safety and efficacy,less side effects,better synergy and lower cost over traditional treatments for cancers.Upconversion nanoparticles?UCNPs?are widely used in PDT triggered by near-infra red?NIR?light since they can convert NIR light with greater tissue penetration depth into short wavelength light that can be absorbed by traditional photosensitizers.UCNPs,as the light transducers and carriers of photosensitizers,have solved a series of problems such as insufficient depth of tumor treatment,poor selectivity of ideal photosensitizers and so on.However,clinical applications of PDT are highly limited due to its low tumor killing efficiency.PDT effects mainly depends on the amount of reactive oxygen species?ROS?.ROS are products of the photosensitive reactions between environmental oxygen and photosensitizer drugs which absorb the UC emission energy under NIR light excitation.Therefore,improvement of PDT efficiency can be achieved by enhancing the fluorescence intensity of UCNPs and/or environmental oxygen level in tumor tissues.However,the tumor microenvironment presents a hypoxic environment,and the oxygen is continuously consumed in the process of PDT,which undoubtedly worsens the PDT effects in turn.Herein,the fluorescence intensity of Gd₂O₃ based UCNPs was enhanced via alkali metal and transition metal ions doping.What's more,the hypoxia problem which limits the clinical applications of PDT was smartly solved by doping Mn ions into the Gd₂O₃ matrix,which not only enhanced the upconversion fluorescence intensity but also endowed O₂ generating capabilities to the Gd₂O₃ based UCNPs.In this thesis,a series of research were conducted aiming at improving the PDT effects via enhancing the fluorescence intensity of UCNPs and solving the tumor hypoxia problem.The main results are as follows:1.Study on preparation,fluorescent properties and applications in tumor PDT of Gd₂O₃:Yb,Tm,Li and Gd₂O₃:Yb,Tm,Zn UCNPs.?1?Firstly,precursor of Gd₂O₃ was prepared by a hydrothermal method,which was further sintered at 900? to obtain Gd₂O₃:Yb,Tm,Li and Gd₂O₃:Yb,Tm,Zn UCNPs.Li⁺/Zn²⁺ ions were selected as co-dopants of Yb³⁺and Tm³⁺in Gd₂O₃ matrix.Under the excitation of 980 nm laser,both materials emitted blue fluorescence.The upconversion fluorescence intensity of Gd₂O₃ nanoparticles was improved by Li⁺ and Zn²⁺ ions doping with appropriate content.With an optimal doping content of 15 mol%Li⁺ or 10 mol% Zn²⁺,and the fluorescence intensity at 488 nm was enhanced to 9.09- and 2.97-fold of Li⁺/Zn²⁺ free Gd₂O₃:Yb,Tm UCNPs,respectively.?2?Human cervical caner cell line?HeLa cell line?was used as a cell model to evaluate the cytotoxicity of the prepared UCNPs.The results indicated that the cell viability was higher than 85% with UCNPs concentration lower than 500?g/mL,suggesting low cytotoxicity of the materials.In Gd₂O₃:Yb,Tm,Zn-mediated PDT,the photosensitizer of merocyanine 540?MC540? which strongly absorbs the up-converting fluorescence of Gd₂O₃:Yb,Tm,Zn UCNPs was selected as the photosentisizer and loaded onto the UCNPs.The results showed that Gd₂O₃:Yb,Tm,Zn UCNPs with higher fluorescence intensity could achieve better killing effects on HeLa cells.In addition,the viability of HeLa cells dropped from 67% to 49% as the post-time extended from 2 h to12 h.2.Study on preparation,properties and applications in tumor PDT of O₂self-generating Gd₂O₃:Yb,Er,Mn UCNPs.?1?Similarly,asingle componentupconverting-catalyticbifunctional Gd₂O₃:Yb,Er,Mn nanomaterial was designed and prepared via co-doping Mn ions with Yb³⁺ and Er³⁺ions into Gd₂O₃ matrix.Under the excitation of 980 nm laser,Gd₂O₃:Yb,Er,Mn UCNPs emitted green and red fluorescence at 540 nm and 663 nm,respectively,which were attributed to the ⁴S_3/2?⁴I_15/2 and ⁴F_9/2?⁴I_15/2 transition of Er³⁺.Doping of Mn ions with an appropriate concent enhanced the up-conversion fluorescence intensity of Gd₂O₃ nanoparticles.With an optimal doping amount of 0.15 mol%,the green and red fluorescence intensity was respectively enhanced to 5.83- and 2.03-fold of Mn-free Gd₂O₃:Yb,Er UCNPs.In addition,the ratio of red to green fluorescence intensity gradually increased from 0.41 to 2.53 with the increase of Mn ions doping ratio from 0.1mol%to 0.9 mol%.?2?In the Na₂HPO₄-citric acid buffer solution with a pH of 5.6⁷.0,the catalytic activities of the Gd₂O₃:Yb,Er,Mn UCNPs toward H₂O₂ decomposition was studied.The results showed that Gd₂O₃:Yb,Er,Mn could accelerated O₂ generation from H₂O₂ in acidic environment.The O₂ production rate gradually increased as the pH value decreased from7.0 to 5.6.Furthermore,the more the Mn ions,the higher the catalytic activity of the UCNPs.?3?The low cytotoxicity and good blood compatibility of the UCNPs were confirmd by MTT method and in vitro hemolysis experiment,respectively.HeLa cells were used to evalute the cytotoxicity of the material.The results indicated that the cell viability was higher than 85% with UCNPs concentration lower than 100?g?mL^-1,suggesting the low cytotoxicity of the materials.?4?Methylene blue?MB?which strongly absorbs the up-converting fluorescence of Gd₂O₃:Yb,Er,Mn was selected as a photosentisizer.In vitro PDT effects were investigated by using HeLa cell line as a cancer cell model.The results showed that cancer cell killing efficiency was increased by 2.35 times via using Gd₂O₃:Yb,Er,Mn instead of Gd₂O₃:Yb,Er with assistance of exogenous H₂O₂ in safe dosage.ROS level in HeLa cells significantly increased while mitochondrial membrane potential remarkably decreased.Results of investigation on subcellular structure demonstrated that an apoptosis followed by secondary necrosis was the primary cell death mode of HeLa cells involved in the Gd₂O₃:Yb,Er,Mn mediated PDT.