Controllable Preparation Of Chiral Molybdenum Oxide Nanomaterial System And Its Therapeutic Mechanism In The Cutaneous Squamous Cell Carcinoma

Objective:Cutaneous squamous cell carcinoma（CSCC）is the second most common cutaneous malignancy after basal cell carcinoma.At present,surgery is the main treatment,however,limited by age and skin lesion,some patients cannot choose surgery.Therefore,photo-responsive therapy becomes an ideal choice.In recent years,functional inorganic optical nanomaterials,as novel agents,have attracted much attention for their low cytotoxicity,easy to be endocytosed and optical conversion effects.However,due to the complexity of tumor microenvironment,traditional nanomaterials still face many challenges in clinical applications.For example,high energy consumption,the long-term toxicity of nanomaterials accumulation,instability in the physiological environment and the tumor metastasis and recurrence after treatment.The introduction of chirality into traditional nanomaterials can provide new insights into the above problems.Firstly,the ligand-inorganic nanoparticle structure coated by chiral amino acid molecules has small size and easy to be endocytosed,which can enhance the stability and dispersion in the physiological environment,further improve the therapeutic performance,and increase the retention time in the tumor tissue through enhanced permeability and retention（EPR）effect.Secondly,chiral nanomaterials exhibit satisfying biocompatibility.The coated amino acid and the specific chirality significantly reduce the in vivo toxicity.Thirdly,the different absorption to different chiral circular polarized light（CPL）is helpful to selective drug-activation in the tumor regional treatment,which improves the treatment efficiency while reducing the energy consumption.Based on this,chiral inorganic nanoparticles（NPs）,including photothermal therapy（PTT）,photodynamic therapy（PDT）and photoacoustic imaging（PAI）,have become a new hotspot in the field of cancer treatment.As a transition metal element,molybdenum has rich valence states and can form different oxygen-deficient structures with oxygen(Mo O_3-x,1<x<3).Due to the oxygen-deficiency and the interaction with chiral ligands on the particle surface,the stoichiometry can be adjusted.The carrier concentration of Mo O_3-xnanoparticles increases during this process,and the surface plasmon absorption appears in the near infrared light（NIR）region.At the same time,Mo O_3-x can not only produce the absorption in the visible region,but also transfer the chirality of the ligand molecules to the above two regions,and form strong chiral absorption peaks.Mo O_3-x NPs can be regarded as candidates for cancer therapy for the adjustable chiral optical absorption properties in the visible light-NIR region.It can improve the energy utilization efficiency through its chiral selective optical activity,save the energy consumption and improve the therapeutic efficiency.Therefore,we intend to synthesize 532 nm visible light and 808 nm NIR dual-channel responsive chiral molybdenum oxide nanomaterials through chiral ligand induction method,then we discuss the potential of the application to multimodal photo-responsive diagnosis and therapy of CSCC,and the mechanism of tumor ablation.Methods:1.Preparation and performance evaluation of chiral molybdenum oxide nanomaterialsBased on the hydrothermal method,the mother liquor of Mo O₃ was obtained by redox reaction of Mo S₂.Then we adjusted the dosage of L-/D-cysteine（L-/D-Cys）to induce the chirality from amino acid ligands to NPs through the interaction of sulfhydryl group（-SH）and transition metal Mo atom,to obtain the controllable chiral optical properties,L-/D-Cys-Mo O₂ and L-/D-Cys-Mo O_2.8 NPs were stably prepared.By controlling the dosage of cysteine molecules（5mg,60mg）,and conducting the chirality induction and redox reaction of high valence oxide of molybdenum,we realized the controllable synthesis of chiral molybdenum oxide NPs with different valence states(L-/D-Cys-Mo O₂and L-/D-Cys-Mo O_2.8 NPs).Then we used X-ray photoelectron spectroscopy（XPS）,transmission electron microscope（TEM）,circular dichroism spectrum（CD）and UV-visible spectrum（UV-Vis）to characterize the morphology,valence states and chirality.The photo-thermal conversion capacity was determined by a temperature sensing device and infrared imaging system.The cellular uptake was observed using TEM.2.The study of biological effects of chiral molybdenum oxide nanomaterials（1）The cell safety concentration of L-/D-Cys-Mo O_3-x（1<x<3）NPs was determined by live/dead staining and CCK-8 test.The residues and excretion,metabolic pathways and rates,toxicity and biocompatibility of molybdenum were assessed by inductively coupled plasma mass spectrometry（ICP-MS）research of the major organs of research animals.The infrared imaging system,reactive oxygen species（ROS）fluorescence probe,photoacoustic imaging system,hematoxylin-eosin（H&E）staining were used to investigate the photothermal effect,photodynamic effect and photoacoustic imaging ability of chiral molybdenum oxide nanomaterials,the ability.The capability,safety and potential of L-/D-Cys-Mo O_3-x NPs guided photo-responsive therapy were confirmed by the results of both in vivo and in vitro tumor chiral photo-responsive therapy.3.The study of the mechanism of tumor ablation during chiral molybdenum oxide nanomaterials mediated photo-responsive therapy of CSCC（1）The apoptosis and autophagy of tumor cells in the treated tissue were evaluated by immunohistochemistry and immunofluorescence.（2）The activation of proteins related to apoptosis and autophagy key signaling pathways and related signaling pathways as well as cascaded reaction pathways in the process of photo-responsive therapy were clarified.Results:1.We successfully prepared the chiral molybdenum oxide nanomaterials with visible light-NIR dual channel chiroptical activity,simultaneous metal-to-ligand charge transfer（MLCT）and surface plasmonic resonance（SPR）chirality and the g-factor larger than traditional materials.L-/D-Cys-Mo O₂ has 532nm photo-responsive activity,and L-/D-Cys-Mo O_2.8 has 532nm photo-responsive activity.This nanomaterial system can provide research foundation for photo-responsive diagnosis and therapy of CSCC;2.The PTT-PDT combination effect mediated by the response at 532 nm of L-/D-Cys-Mo O₂ can induce the apoptosis and autophagy of tumor cells,which enhanced the photo-responsive therapeutic effect and achieved the high-efficiency tumor ablation.Moreover,the induction of chirality realized the selective absorption of left/right CPL,which further improved the efficiency of tumor therapy,saved the energy consumption and made the L-/D-Cys-Mo O₂ into a candidate for the new generation of CSCC photo-responsive therapeutic substrate;3.L-/D-Cys-Mo O_2.8 has 808 nm NIR response ability.It guided tumor ablation through PTT-based photothermal conversion effect and photoacoustic imaging with tissue penetration ability,and showed a‘tumor suppression effect’of one photo-responsive therapy and several days of reduction.The main mechanism is that the mitochondria-mediated apoptosis signaling pathway is cascade activated and induces the apoptosis of tumor cells;4.Molybdenum is one of the essential trace elements in animals,which shows high safety.The nano-scale chiral molybdenum oxide obtained by the chiral amino acid coating technology shows higher biocompatibility.The animal level experiment results showed that the chiral molybdenum oxide nanomaterial injected intravenously can be mostly excreted through the kidney within 7 days.Conclusions:1.In terms of technics,a chiral molybdenum oxide nanomaterial system with good biocompatibility,dispersibility,and visible light and near-infrared light dual-channel response ability was obtained;2.In terms of application,based on the 532 nm visible light and 808 nm NIR dual-responsive properties of chiral molybdenum oxide NPs,the effect of PTT,PDT and PAI for the diagnosis and therapy of CSCC was confirmed;3.In terms of mechanism,the biological mechanism of inducing tumor cells’apoptosis was reveled,which is the chiral molybdenum oxide NPs stimulate the cascade reaction of mitochondrial apoptosis through photo-conversion.This mild and effective therapy has the good prospect of clinical transformation.