Liposome Structure Regulates The Optical Properties Of Cyanine Dyes And Its NIR-Ⅱ Imaging And Treatment

Near-infrared two-region（NIR-Ⅱ）fluorescence imaging（FI）,as a novel type of clinical imaging method,has attracted more attention due to its high temporal resolution and sensitivity imaging performance in recent years.Numerous studies have shown that loading NIR-Ⅱ fluorophores into nanomaterials can effectively improve their biocompatibility,avoid solvent quenching effects,and increase their targeting function.Moreover,there are also studies indicating that the nano-scale space of nanomaterials will induce NIR-Ⅱ fluorophores to change their structure,thereby altering their fluorescence properties.NIR-Ⅱ fluorescence materials with unique fluorescence properties can achieve a wider range of biological applications.However,there is no in-depth and systematic study on the molecular structure and spatial distribution of NIR-Ⅱ fluorophores in nanomaterials,as well as their impact on their optical properties.The unique bilayer membrane spatial structure of liposomes（LPs）makes it easier to achieve changes in the structure and performance of fluorophores.In this study,the interaction between NIR-Ⅱ fluorophores and nanomaterials was studied based on the structure of liposomes.Fluorescent liposomes with special optical properties were obtained by studying the fluorescence properties of various liposomes loaded with cyanine dyes IR-1061 and indocyanine green（ICG）.Then,a universal theoretical basis for the design and research of functionalized cyanine dye liposomes were established by in-depth researching on the molecular mechanisms of the optical properties of these fluorescent liposomes through molecular simulation.Finally,we utilized the reasonably designed liposome nanoplatforms to establish two fluorescent probes,one of which was able to get excellent NIR-Ⅱ imaging performance from NIR-I fluorophore ICG,the other is based on the fluorescence regulation of NIR-Ⅱ fluorophore IR-1061 to achieve multifunctional diagnosis and treatment integration of imaging and thermochemotherapy.The research content includes the following three parts:（1）Construction of J-aggregated ICG liposomes to achieve NIR-Ⅱ FI performance of NIR-I cyanine fluorophoreLiposomes with different phospholipid loaded with ICG were prepared and their physicochemical properties were analyzed.Aqueous cationic（DOTAP）liposomes（ICG@CLPs）with an average particle size of 158.9 nm and an encapsulation efficiency of 90.3%were prepared.Spectral analysis results showed that the absorption spectra of ICG@CLPs shifted from 780 nm to 890 nm,achieving a wavelength red-shift of 110 nm;The corresponding emission spectra shifted from 830 nm to 920 nm,achieving a wavelength red shift of 90 nm,with tail peaks exceeding 1000 nm.However,ICG@CLPs will gradually disappear with the increase of ICG drug loading.The results showed that ICG@CLP-3（with a drug loading of 3%）possessed the maximum FI performance and good NIR-Ⅱ FI effect.ICG@CLP-3 liposomes modified with IF₇ peptide（IF-ICG@CLP-3）were further prepared and evaluated for in vivo NIR-Ⅱ FI.Angiographic results in mice demonstrated that IF-ICG@CLP-3 could display microvessels that could not be displayed by NIR-I FI in the abdomen of mice,which demonstrated that IF-ICG@CLP-3 had good tissue spatial resolution.Imaging of subcutaneous transplanted tumors in mice demonstrated that IF-ICG@CLP-3 could effectively target mouse tumors within 4 hours.The signal intensity of tumors was 1.5 times that of ICG@CLP-3,which demonstrated that IF-ICG-CLP-3 had good tumor-targeting ability.In order to explore the molecular mechanism of ICG@CLPs structure causing spectral redshift of ICG molecules,the molecular dynamics simulation models of ICG molecules and DOTAP molecules were further established to elucidate the structural changes of ICG molecules in liposomes and the causes.The effects of ICG concentration and distribution in lipid membranes on the structure and spatial distribution of ICG were analyzed.The results showed that the electrostatic interaction of ICG molecules in the inside system（ICG in the phospholipid bilayer）increased their distribution space in the liposome,so they did not exhibit aggregated state.However,the ICG molecules in the outside system（ICG outside the phospholipid bilayer）exhibited face-to-face dislocation stacking（J-aggregated state）due to both electrostatic and hydrophobic interactions.The results demonstrated that the spectral redshift of ICG@CLPs is due to the J-aggregation of ICG.（2）Construction of H-aggregated IR-1061 liposomes to enhance the photothermal properties of NIR-Ⅱ fluorophoreLiposomes with different phospholipid loaded with NIR-Ⅱ cyanine dye,IR-1061,were prepared.Their physicochemical properties were analyzed and their formulations were optimized to establish the optimal lipid preparation scheme.IR-1061 anionic liposomes（IR-ALPs）with special optical properties were obtained by further analysis and research on the optical and photothermal properties.IR-ALPs exhibited a significant blue shift in their absorption spectra as the drug loading of IR-1061 increased,resulting in a decrease in fluorescence performance and an improvement in photothermal performance.Through reasonable design,we obtained the IR-1061 liposome IR-ALP-4（with a drug loading of 2%）that maximizes fluorescence intensity and good photothermal performance.The photothermal conversion efficiency of IR-ALP-4 reaches 42%,which can simultaneously achieve dual functions of NIR-Ⅱ FI and NIR-I photothermal therapy（PTT）.In order to explore the molecular mechanism of IR-ALPs structure resulting in enhanced photothermal performance of IR-1061 molecule,the molecular dynamics simulation models of IR-1061 molecule and DPPG molecule were further established to elucidate the structural changes of IR-1061 molecule in liposomes and the causes.The structure and spatial distribution of IR-1061 in phospholipid bilayer at different concentrations were analyzed and the results showed that IR-1061 molecule was affected by nano-scale space in phospholipid bilayer.With the increase of concentration,the spatial distribution of IR-1061molecule changed from single-layer disordered distribution to multilayer overlapping stacking distribution.When the ratio of IR-1061 molecule to DPPG molecule reached 1:5,66%of IR-1061 molecule formed a face-to-face overlapping stacking state（H-aggregated state）,which implemented the transition of IR-1061from free state to H-aggregated state in liposomes.The results demonstrated that the enhanced photothermal properties of IR-ALPs are due to the H-aggregates of IR-1061.（3）Integration of diagnosis and treatment of non-small cell lung cancer based on H-aggregated fluorescent liposomes with membrane delivery functionA multifunctional fluorescent probe（RR-IR-ALP-C）with surface modification of RR₉ peptides and encapsulation of Carpolatin was designed and prepared based on IR-ALP-4 liposome.The results indicated that carrying IR-1061 in DPPG liposomes results in the simultaneous presence of NIR-Ⅱ IF performance endowed by the free state of IR-1061 and NIR-I PTT performance endowed by the H-aggregated state of IR-1061.Modifying RR₉ peptides on the surface of RR-IR-ALP-C increased the ability of liposomes to be uptaked by tumor cells through the enhanced mechanism of membrane fusion,which was able to transfer IR-1061 with H-aggregated state onto cell membranes simultaneously,thereby maintaining PTT capacity for more than 12 hours.The thermosensitive drug releasing and the combined therapeutics of thermo-chemotherapy increased the killing ability of RR-IR-ALP-C on A549 cell.The results showed that RR-IR-ALP-C could kill 55.3%of A549 cell,much higher than the group of carboplatin alone（30.6%）and thermotherapy alone（37.8%）.Animal experiments have shown that RR-IR-ALP-C can achieve both high signal tumor NIR-Ⅱ FI and photothermal imaging（PTI）to achieve bimodal imaging.RR-IR-ALP-C can also achieve effective treatment of tumors through the combination of thermosensitive drug release and thermochemotherapy.The treatment results of subcutaneous transplantation tumors of A549 cells indicated that RR-IR-ALP-C can effectively reduce the volume of tumor tissue from 100 mm³ to about 60 mm³ after 16 days of irradiation,which is much smaller than the group of carboplatin alone group（230 mm³）and the hyperthermia alone（290 mm³）.