The Study Of ?-NaREF₄:Ln³⁺-Based Nanoparticle-Photosensitizer Nanocomposites For X-Ray Excited Photodynamic Therapy Application

As an alternative way of cancer treatment,photodynamic therapy?PDT?has the advantages of non-invasive,neglectable side effect and controllable cytotoxicity in the specific area.The key factors of PDT are excitation light,oxygen,and photosensitizer?PS?.The PS is stimulated by excitation light to react with nearby oxygen to generate excited-state singlet oxygen?SO?,which could efficiently oxidize the biological molecules,such as aliphatic acid,protein,nucleic acid,and mitochondrial membrane,to induce cell death.However,due to the limited penetration of UV-visible light in tissues,PDT is generally used to treat lesions located superficially or being reachable by optical fibers,such as skin cancer,gastrointestinal tumor,respiratory tumor,and genitourinary tumor.Recent years,the near-infrared?NIR?light excited PDT based on luminescent nanoparticles?NPs?such as upconversion nanoparticles?UCNPs?,quantum dots?QDs?,carbon nanomaterials?CNMs?,and gold nanomaterials?GNMs?,can extend penetration depth and have gained great attraction in cancer therapy.Though these NPs can absorb energy from external NIR source and generate UV/visible light to excite PS,the penetration depth of NIR light in tissues is still limited,which is less than 1.5 cm to maintain enough energy for PDT.Effective PDT for deep-seated or larger tumors is still a great challenge.Using X-ray as the excitation source,which can achieve unlimited penetration depth in vivo,has the ability to overcome the penetration depth issue.However,clinically-used PS cannot be directly stimulated by X-ray.Thus,an energy transducer,such as X-ray excited luminescent nanoparticles?XLNPs?,is needed to transfer energy from X-ray to PS.The X-ray excited photodynamic therapy?X-PDT?modality has been proposed.X-ray excited luminescent nanoparticle-photosensitizer nanocomposites can increase the generation rate of reactive oxygen species?ROS?,and improve the tumor treatment effect.Nevertheless,in current studies of X-PDT,high-dose X-ray radiation,usually between 2-8Gy reported in the literatures,was used to achieve moderate treatment efficacy.To enable X-PDT for clinical cancer treatment,high luminescent yield of NPs,high fluorescence resonant energy transfer?FRET?efficiency of the nanoparticles-photosensitizer?NPs-PS?nanocomposite,and effective endocytosis of NPs-PS are the main factors to lower X-ray radiation dosage.Synthesizing XLNPs with strong luminescence and perfectly matched spectrum with PS will not only increase the FRET efficiency but also boost up the SO generation for X-PDT.Synthesis of XLNPs with suitable size,which is less than 100 nm,can efficiently enhance the enrichment of NPs-PS in tumor sites by the enhanced permeability and retention?EPR?effect.Therefore,to achieve the aims mentioned above,four parts of the study contents have been concluded in this research.Part?Synthesis of OA-capped XLNPs with strong luminescence and perfectly matched spectrum with PS.To solve those issues of large size,uneven particle size,irregular shape,weak light emission of XLNPs,OA-stabilized XLNPs were synthesized via a modified co-precipitation routine which was adopted from Nature Protocol.Those XLNPs contained?-NaGdF₄:X%Tb³⁺NPs?X=3,5,7,12,15,18,22 mol%?,NaLuF₄:X%Tb³⁺NPs?X=3,6,9,12,15,18,21 mol%?,and NaLuF₄:X%Gd³⁺15%Eu³⁺NPs?X=5,10,15,20,25,30 mol%?.We studied the reaction mechanism,luminescence intensity,particle size and morphology control in each synthesis process by changing the reaction temperature,reaction time,doping ratio of the luminescent center,and the molar ratio of the fluorine source and the sodium source.The X-ray excited luminescent spectrum of such XLNPs was characterized by the fluorescent spectrometer,and morphological characterization was characterized by TEM,HR-TEM,SEM,XRD,EDS element mapping.The size of?-NaGdF₄:15%Tb³⁺NPs was about 9 nm with strong green fluorescence,but the X-ray excited luminescent spectrum of?-NaLuF₄:15%Tb³⁺NPs with larger size was almost seven times higher than that of?-NaGdF₄:15%Tb³⁺NPs at545 nm peak.Plus,the X-ray excited emission spectrum of?-NaREF₄:15%Tb³⁺NPs?RE=Gd,Lu?matched well the absorption spectrum of PS named rose bengal?RB?.In addition,the X-ray excited luminescence intensity of NaLuF₄:X%Gd³⁺15%Eu³⁺NPs?X=5,10,15,20,25,30 mol%?NPs was low,and the characteristic emission peak of Eu³⁺was not matched with the absorption spectrum of RB.Hence,the?-NaREF₄:15%Tb³⁺NPs?RE=Gd,Lu?were used for the following research.Part?Synthesis and the singlet oxygen quantification of the?-NaREF₄:15%Tb³⁺NPs-RB?RE=Gd,Lu?nanocomposite.Unfortunately,the as-synthesized?-NaREF₄:15%Tb³⁺NPs?RE=Gd,Lu?could not be dispersed in water and not suitable for biomedical applications,because such NPs were wrapped with oleic acid?OA?groups,which were hydrophobic.2-aminoethylphosphonic acid?AEP?was employed to modify the NPs surface by a ligand-exchanged process.The successful ligand-exchange with AEP was confirmed via Fourier-transform infrared spectroscopy?FT-IR?.Plus,the excitation spectrum of?-NaREF₄:15%Tb³⁺NPs?RE=Gd,Lu?was perfectly matched with the absorption spectrum of RB at 545 nm.The covalent conjugation of NPs-RB was adopted to ensure the distance between the luminescent center?donor?and the RB?acceptor?close enough for the high FRET efficiency.N-?3-dimethylaminopropyl?-n?-ethylcarbodiimide hydrochloride?EDC?were used to enable the covalent conjugation between-COOH and-NH₂ groups.With the ratio of NPs:RB?w/w?at 20:1 and 19.53125:1,these nanocomposites showed the maximum of FRET efficiency,which were 99.63%and94.29%for?-NaGdF₄:15%Tb³⁺NPs@AEP-RB and?-NaLuF₄:15%Tb³⁺NPs@AEP-RB,respectively.Generation of SO were detected and quantified chemically via 1,3-diphenylisobenzofuran?DPBF?.Upon X-ray radiation,the SO quantum yield of NPs-RB has a linear relationship with the radiation time.Corresponding with FRET efficiency,the higher of FRET efficiency,more SO was generated in the unit of time.Part?Evaluation of in vitro X-PDT efficacy.To evaluate the in vitro biosafety of X-ray excited luminescent nanoparticle-photosensitizer nanocomposites,the endocytic efficiency by EPR effect,and the curative effect of in vitro X-PDT,the in vitro X-PDT performance of NPs-RB was evaluated via 3-?4,5-dimethyl-2-thiazolyl?-2,5-diphenyl-2h-tetrazolium bromide?MTT?and cell counting kit-8?CCK-8?assays.HepG2 cells were incubated with?-NaREF₄:15%Tb³⁺NPs@AEP and?-NaREF₄:15%Tb³⁺NPs@AEP-RB?RE=Gd,Lu?nanocomposite for different time?0,2,4,6,8,10,12 h?,and these results implied that the NPs and NPs-RB were water soluble and non-cytotoxic at the concentration up to 1 mg/mL.To further illustrate the influence of irradiation dosage and NPs-RB concentration on X-PDT efficacy,the cell viability of HepG2 cells was systematically studied under different combination of these factors.When X-ray irradiated for 1.5 h,the cell death rate of?-NaREF₄:15%Tb³⁺NPs@AEP-RB?RE=Gd,Lu?nanocomposite were respectively 80%?RE=Gd?and 90%?RE=Lu?.In addition,in vitro SO generation was confirmed by using 2?,7?-dichlorofluorescin diacetate?DCFH-DA?,which was sensitive to SO and generated green fluorescence?Ex/Em 488/525?upon the presence of SO.For NPs-RB with 1.5 h X-ray irradiation strong green fluorescence from DCFH-DA could be observed in the whole cell region,confirmed that the reason of cell death was the generation of SO by X-PDT.Those results laid the foundation for in vivo cancer treatment experiments.Part?Evaluation of in vivo X-PDT efficacy.To evaluate the in vivo biosafety of X-ray excited luminescent nanoparticle-photosensitizer nanocomposites,and the curative effect of in vivo X-PDT,the in vivo X-PDT efficacy of NPs-RB was assessed by xenograft mouse models via subcutaneous injection.All results of?-NaGdF₄:15%Tb³⁺NPs@AEP-RB nanocomposite showed that with a relatively low dosage?0.25 Gy each time,X-ray irradiation four times,total 1 Gy?,the average antitumor efficiency was 60%.In addition,with a single time of 20 min X-ray irradiation,the?-NaLuF₄:15%Tb³⁺NPs@AEP-RB nanocomposite could achieve partial antitumor efficiency?80%±12.3%?with minimal side effect at low X-ray dosage?total 0.19 Gy?.The results from TUNEL assay indicated that there were HepG2 cell apoptosis and a large area of necrosis in the group of?-NaREF₄:15%Tb³⁺NPs@AEP-RB?RE=Gd,Lu?nanocomposite after X-ray irradiation.The H&E stain proved that the side effect?body weight decreasing?was mainly from X-ray but not?-NaREF₄:15%Tb³⁺NPs@AEP-RB?RE=Gd,Lu?nanocomposites.The H&E staining of major organs in each group without X-ray irradiation and the group with X-ray irradiation for 20 min?0.19 Gy?were in good condition.However,in the other groups with X-ray irradiation,the certain damage occurred in the major organs.Based on the results of four research contents,the main conclusions and novelties are as follows:Firstly,with the successful synthesis of?-NaREF₄:15%Tb³⁺NPs?RE=Gd,Lu?,those NPs were uniform spherical morphology,uniform particle size distribution,and monodispersity by the improved co-precipitation method.The average sizes of NPs were about 9 nm and 28 nm,respectively,which could enhance the EPR effect and increase the cell endocytic efficiency,so that the XLNPs could concentrate at the tumor site.Furthermore,the highest peak value of X-ray excited luminescent spectrum of those NPs was at 545 nm,which matched well with the absorption spectrum of RB,and the luminescence yield was high.The highly efficient luminescent core was provided for the synthesis of X-ray excited luminescent nanoparticle-photosensitizer nanocomposites.Secondly,to obtain dispersity in aqueous solutions,the ligand-exchanged process was performed to modify the?-NaREF₄:15%Tb³⁺NPs?RE=Gd,Lu?surface with amino group by using AEP,and confirmed via FT-IR.Such NPs covered with AEP were ready for PS conjugation as well as increasing biocompatibility.The simple and efficient?-NaREF₄:15%Tb³⁺NPs?RE=Gd,Lu?@AEP-RB nanocomposite were prepared via covalent conjugation.The high FRET efficiency of those nanocomposite were 99.63%(?-NaGdF₄:15%Tb³⁺NPs@AEP-RB)and 94.29%(?-NaLuF₄:15%Tb³⁺NPs@AEP-RB).Thirdly,treatment groups using different nanocomposite with low dose X-ray irradiation,the survival rate of HepG2 cell were 20%(?-NaGdF₄:15%Tb³⁺NPs@AEP-RB)and 10%(?-NaLuF₄:15%Tb³⁺NPs@AEP-RB),respectively.Fourthly,based on the nude mouse of heterotopic tumor transplantation,the experiment results showed that the average tumor inhibition rate of the?-NaGdF₄:15%Tb³⁺NPs@AEP-RB nanocomposite treatment group was 60%with total 1 Gy X-ray irradiation,while the?-NaLuF₄:15%Tb³⁺NPs@AEP-RB nanocomposite treatment groups could reach 80%±12.3%with only 0.19 Gy X-ray irradiation.