Ultralong-circulating And Self-targeting "Watson-Crick A= T"-inspired Supramolecular Nanotheranostics For NIR-Ⅱ Imaging-guided Synergistic Photothermal-chemotherapy

A carrier-free nanodrug directly co-assembled using a NIR probe and a chemotherapeutic drug is a hot research topic in cancer therapy.Nevertheless,carrierfree nanodrugs still face two key scientific problems to be solved:short circulation time in vivo and poor accumulation/tumor cell uptake at the tumor site.In recent years,surface functionalization with polyethylene glycol(PEG)and exogenous targeting ligands to prolong blood circulation time,improve tumor site accumulation,and enhance tumor cell uptake efficiency has been extensively investigated.However,most exogenous targeting ligands and PEG have no therapeutic effect.Here,we designed an ultralong circulation and self-targeting nanotheranostics based on supramolecular chemistry through the ordered co-assembly between three FDA-approved indocyanine green(ICG,a near-infrared fluorescent probe),methotrexate(MTX,a folic acid-like antitumor drug that inhibits dihydrofolate reductase),and clofarabine(CA,a purine nucleoside derivative that inhibits nucleotide reductase).First,MTX and ICG were coassembled into the semi-assembled ICG-MTX i.v.multiple weak interactions,the supramolecular co-assembly strategy that not only achieve ultra-high drug payload,but also realize the functions of self-targeting,diagnostics,and synergistic photothermalchemotherapy.Subsequently,a surfactant-like chemotherapeutic drug CA due to its double hydroxyl structure and aromatic ring backbone was introduced into the initial semi-assembled ICG-MTX via "Watson-Crick A=T-inspired" hydrogen bond-driven precise assembly with MTX to result in the formation of supramolecular nanotheranostics(CA@ICG-MTX).The details of the paper are as follows:(1)Synthesis and characterization of CA@ICG-MTX.CA@ICG-MTX was constructed via an assembly/interface-disassembly/secondary-assembly method between the self-targeting chemotherapeutic drug MTX,surfactant-like chemotherapeutic drug CA,and photosensitizer ICG.The hydrodynamic diameter,polydispersity index(PDI)and zeta potential were measured by dynamic light scattering(DLS)and electrophoretic light scattering(ELS).Morphology was observed by scanning electron microscopy(SEM)and transmission electron microscopy(TEM).The successful synthesis of CA@ICG-MTX was verified using nuclear magnetic(1H NMR)spectroscopy,X-ray diffraction(XRD)spectroscopy,fourier transform infrared(FT-IR)spectroscopy,ultraviolet-visible-near infrared(UV-vis-NIR)absorption spectroscopy and fluorescence spectroscopy.The results showed that the size of CA@ICG-MTX MTX is about 131.0 nm,the distribution is uniform,and showed a spherical shape with a drug payload of 100 wt%and successfully verified the existence of multiple weak interactions between MTX,ICG and CA.(2)Stability,photothermal conversion performance,PA/NIR-Ⅱ fluorescence imaging capability,and stimulus-responsive ability of CA@ICG-MTX were investigated.The stability of CA@ICG-MTX was verified by observing the changes of size,potential,absorption and fluorescence intensity of CA@ICG-MTX by DLS,ELS,UV-vis-NIR absorption spectra and fluorescence spectra over 7 days.The in vitro photothermal performance of CA@ICG-MTX after laser irradiation was monitored by infrared camera to evaluate its effect on the photothermal conversion efficiency of ICG.Then to examine the ability of CA@ICG-MTX as a contrast agent for PA/NIR-Ⅱfluorescence imaging,the imaging signal was measured and quantified.Finally,the responsiveness of CA@ICG-MTX to acidity and external laser stimulation was explored.The results showed that CA@ICG-MTX not only exhibit excellent stability,photothermal conversion ability,and photoacoustic/NIR-Ⅱ fluorescence performance,but also exhibit strong acidity/laser dual stimulation response ability.(3)In vitro and in vivo anti-tumor performance study of CA@ICG-MTX.The specific self-targeting ability of CA@ICG-MTX was studied qualitatively by cellular uptake assay using confocal laser scanning microscopy(CLSM),and then the in vitro antitumor activity of CA@ICG-MTX was investigated using standard MTT methods.The cell lines used for the cell experiments included folate receptor-positive 4T1 and HeLa cells and folate receptor-negative A549 cells.Their self-targeting ability in vivo was then analyzed by NIR-Ⅰ/NIR-Ⅱ fluorescence imaging and PA imaging techniques,and the optimal time point for photothermal treatment was determined.Finally,the in vivo antitumor performance of CA@ICG-MTX was validated using a 4T1 tumor nude mouse model and its biosafety was examined.The results showed that CA@ICG-MTX not only has superior physiological stability compared to ICG-MTX,but also possesses an ultra-long blood circulation time(>7 days)and is able to accumulate efficiently at tumor sites in vivo.In addition,CA@ICG-MTX can be specifically recognized by tumor cells with high expression of folate receptors and release the drug on demand under the dual stimulation of lysosomal acidity and external laser.The synergistic photothermal therapy and chemotherapy,guided by NIR-Ⅱ fluorescence imaging,can achieve efficient tumor ablation in one treatment cycle while preventing tumor recurrence with no significant toxic side effects.Therefore,our ultralong-circulating and self-recognizing carrier-free theranostic nanodrug based on the "drug-deliveringdrug" strategy might have the potential for clinical theranostic application.