Effect Of Hydrophilic Macromolecule Delivery Platform On The Performance Of Visible/Near Infrared Ⅱ Fluorescent Small Molecular Antitumor Theranostic Agents

Diagnosis and therapy are two of the most important parts of cancer treatment.Mitochondria targeting delocalized lipophilic cation（DLC）5BMF shows great selectivity and cytotoxicity toward tumor cells,and because of its conjugation backbones,5BMF has intrinsic fluorescence,making it possible as multifunctional theranostic anticancer agents combining targeting,therapy and imaging.However,its high lipophilicity limits the in vivo bio-availability after administration.Usually,covalent modification by hydrophilic groups would increase DLC’s solubility,but it could also lower these molecules’permeability across bio-membranes,complicate the preparing process as well as hamper further large-scale production for clinical application,therefore a simple and convenient in vivo delivery vehicle is required.As endogenous protein,human serum albumin（HSA）has good solubility and small molecule delivery capacity with simple loading methods,which is quite suitable for 5BMF.On the other hand,the fluorescence spectra of DLCs mainly fall in the visible band and has shallow penetration depth in bio-tissues with poor in vivo fluorescence imaging outcomes.Recently,near infrared II imaging has been drawing more and more interest for its deeper tissue penetration and higher imaging contrast and resolution.One kind of NIR-II dyes,small molecular dyes（SMDs）,shows rapid in vivo clearance and great biocompatibility,therefore possessing great clinical application potential.However,the current SMDs library is not big enough to cover the whole NIR-II spectrum and existing wavelength tuning methods require complicated chemical modification,hampering novel SMDs developing.Therefore,it would be meaningful to conjure new simple and convenient tuning strategies to facilitate the promotion of SMDs.To address above problems,the following researches have been carried out and discussed in this thesis:（1）.DLC drug 5BMF was synthesized and loaded to HSA via convenient non-covalent binding methods,forming 5BMF@HSA,and its solubility was improved from 1.61 mg/m L to 5.41mg/m L.Around 2 folds enhancement of the fluorescence intensity by HSA was found in the emission spectra,and in fluorescence titration experiment,the binding number of 5BMF to HSA was～1:1.Molecular docking was conducted to explore the binding mechanism which was found to be the electrostatic interaction and like-dissolves-like principle.Also,in docking results the hydrophobic binding site excluded water molecules and other 5BMF around the DLC drug,resulting in the fluorescence enhancement.5BMF@HSA showed selective releasing behavior in different p H environment as more than 10 folds of 5BMF was released in acidic PBS（p H=5.3）compared to neutral and basic solution.（2）In vitro and in vivo experiments were performed to study the imaging ability,cellular localization,and anticancer activity of 5BMF@HSA.Cellular fluorescence imaging indicated that the introduction of HSA altered the intracellular distribution of 5BMF as its signals were observed both in mitochondria and lysosomes,which could be the cause for improved cellular uptake of the drug.Besides,nearly 1.5 folds increase of intracellular fluorescence intensity was detected in the imaging results.5BMF@HSA showed selective cytotoxicity toward cancer cells in proliferation inhibition test where its half inhibitory concentration(IC₅₀)against carcinomatous cells was 23 times as low as that against normal cells.The results of in vivo tumor treatment experiment demonstrated that the complex inhibited tumor growth effectively and its anticancer activity was better than free 5BMF molecules.Above outcomes revealed the potential of 5BMF@HSA being theranostic agents,but the weak tissue penetration ability of its fluorescence emission limited its further application for in vivo imaging.（3）A series of SMDs,namely FFB,FTB and FSB,with different heteroatoms in donor units were designed and synthesized to verify our newly proposed NIR-II fluorescence emission tuning strategy by simple atom alteration.This method could avoid complicated modification of chemical preparation process and is suitable to most of currently reported SMDs as well as the future design of novel NIR-II structures.The fluorescence spectra confirmed the feasibility of this strategy as the emission peak of FFB,FTB and FSB were 991 nm,958 nm and 1033 nm,respectively.The quantum yields were also determined to be 0.70%,0.79%and 0.55%.By density function theory calculations,we found that the underlining wavelength tuning mechanism is the combination results of the electronegativity of the donors’heteroatoms and the 3D structural configuration,and weaker electronegativity and planar structure would force the emission to red-shift,which was instructive for future NIR-II SMDs design.（4）According to the molecular docking simulation and inspiration of 5BMF@HSA results,HSA was used as delivery vehicle to improve the aqueous solubility and fluorescence intensities of FFBA,FTBA and FSBA（A represented carboxylic acid group）.Convenient non-covalent binding method was also employed here to prepare these SMDs@HSA,and conditional experiment demonstrated that the optimal molar ratio between the small molecules and HSA was 2:1 and heating temperature for preparation was 50^oC,by which the fluorescence intensities in water of FFBA,FTBA and FSBA were improved 41.6,55.4 and 60.5 times,respectively.During in vivo whole body vascular imaging experiment,we found that FFBA@HSA showed the best imaging effect with 1150LP optical filters,and exhibited high signal to background ratio（6.4）and spatial resolution（～400μm）in tumor vascular and lymph node imaging with good stability lasting longer than 24 h,suggesting that this complex has great potential in NIR-II imaging.（5）PEG was introduced to FFBA and formed FFBA-PEG.The new structure showed greatly increased solubility and tumor targeting ability.It clearly outlined tumor boundaries with high imaging contrast and successfully guided the surgical removal of primary tumors as well as small secondary metastatic ones by NIR-II imaging.FFBA-PEG had higher photothermal conversion efficiency than many SMDs reported before and effectively ablated cancerous tissues after photothermal therapy.This work offered a novel small molecular platform to combine imaging and therapy for tumor treatment with better aqueous solubility,imaging quality and photothermal effect.