Study On Two-dimensional Nanosheets(BiOCl And AsP) For Breast Cancer Theranostics

Background:Breast cancer is one of the greatest threats to females’health.Traditional therapeutic methods,i.e.surgery,chemotherapy and radiotherapy are still far from satisfactory.With the fast development of nanotechnology,nanomaterials fabricated by integerating imaging and therapeutic moieties together show great potential in the field of nanomedicine,especially in tumor theranostics.Therefore,rational engineering materials at an atomic or molecular level and exploring novel multifunctional theranostic nanoplatforms are of great importance for highly efficient breast cancer therapy.Objective:To prepare novel multifunctional theronostic nanosheets with intrinsic imaging and therapeutic capabilities into a single nanoframe,which could allow precise PA imaging monitoring and efficiently achieve inhibition of breat tumor growth.Methods:In this study,we elaborately developed and constructed two kinds of 2D theranostic nanosheets,i.e.polyvinyl pyrrolidone（PVP）modified oxygen vacancies-based BiOCl（O-BiOCl-PVP）nanosheets and bovine serum albumin（BSA）modified AsP（AsP-BSA）nanosheets for efficiently PA imaging guided breast cancer treatments.First,a novel multifunctional theranostic O-BiOCl nanosheet was fabricated by a specific defect engineering strategy.The morphology was observed under transmission electron microscopy（TEM）,scanning electron microscopy（SEM）and atomic force microscopy（AFM）.The electron spin resonance（ESR）technique was employed to detect oxygen vacancies on the surface of the O-BiOCl nanosheets.X-ray photoelectron spectroscopy（XPS）was performed to reveal the chemical composition and status of the BiOCl and O-BiOCl nanosheets.A theoretical DFT simulation and UV-visible（UV-vis）diffuse reflectance spectroscopy were performed to investigate the impact of doping on the band gap and the optical properties of O-BiOCl nanosheets.The photothermal performance of O-BiOCl nanosheets were conducted on an infrared thermal imaging instrument and 808 nm high-power multimode pump laser,and the relationship between oxygen vacancy substitution ratios and extinction coefficients and photothermal convention of O-BiOCl nanosheets was further evaluated.To further improve the biocompatibility,PVP was adopted for surface modification,and FTIR spectrum was performed to test the variation of characteristic peaks between O-BiOCl nanosheets and O-BiOCl-PVP nanosheets.The efficacy of O-BiOCl-PVP nanosheets for PA and CT imaging was evaluated by in vitro and in vivo experiment.4T1 tumor-bearing mouse were utilized as animal mode to assess the breast tumor growth inhibition after photonic tumor ablation enabled by O-BiOCl-PVP nanosheets.Second,a distinct 2D black AsP nanosheet was rationally designed and engineered by an alloy strategy followed by BSA surface modification.TEM,SEM and AFM were performed to observe the structure of AsP nanosheets.The photothermal efficiency of AsP nanosheets was assessed on an infrared thermal imaging instrument and 1064 nm high-power multimode pump laser.After BSA surface engineering,CCK-8 protocol and flow cytometric apoptosis assay were carried out to evaluate the biosafety of AsP-BSA nanosheets as compared with free As₂O₃.XPS was performed to analyze the chemical state of elements in AsP-BSA nanosheets before and after laser irradiation.For evaluating in vivo toxicology of AsP-BSA nanosheets,the normal hematological biochemical indexes as well as hematoxylin and eosin（H&E）staining of major organs（heart,liver,spleen,lung and kidney）of all mice in each group were tested after intravenous injection at varied time points of 3,7 and 28 days.The PA imaging performance of AsP-BSA nanosheets was evaluated by in vitro and in vivo experiment.4T1 tumor-bearing mouse were used as animal mode to evaluate the therapeutic efficiency of photonic cancer therapy and enhanced As-based chemotherapy by AsP-BSA nanosheets.Results:For O-BiOCl-PVP nanosheets,the SEM,TEM and AFM images showed a tetragonal structure of O-BiOCl nanosheets.EPR and XPS spectra of the O-BiOCl nanosheets before and after UV-light irradiation showed a successful production of oxygen vacancies on the surface of O-BiOCl nanosheets.The theoretical DFT simulation and UV-vis diffuse reflectance spectroscopy spectra revealed that the oxygen vacancies in the O-BiOCl samples had a significant impact on the band structures and played a key role in enhancing the light absorption ability.The in vitro extinction coefficients and photothermal-conversion efficiencyηof O-BiOCl nanosheets were calculated to be 6.93 Lg^-1cm^-1,13.9%,respectively,both of which followed a line relationship with oxygen vacancy substitution ratios of O-BiOClnanosheets.After PVP surface modification,FTIR spectrum of the O-BiOCl-PVP nanosheets below 2000 cm^-1displayed a similar trend as that for PVP,indicating the successful surface modification of O-BiOCl nanosheets by PVP.For PA imaging,the fabricated O-BiOCl-PVP nanosheets verified that as the amount of oxygen vacancies increased,the PA signal intensity of O-BiOCl-PVP nanosheets correspondingly enhanced.These nanosheets displayed intriguing CT imaging capability because of strong Bi-based X-ray attenuation coefficient and the in vitro results exhibited that significantly enhanced hounsfield unit（HU）values were observed with elevated Bi concentrations.The in vivo results demonstrated that O-BiOCl-PVP nanosheets could behave as robust contrast agents for PA/CT imaging and efficiently achieve the photothermal inhibition of 4T1 breast tumors.As for AsP nanosheets,the SEM,TEM and AFM images exhibited the planar topology of AsP nanosheets.The in vitro extinction coefficients and photothermal-conversion efficiencyηof the obtained AsP nanosheets were calculated to be 9.39 Lg^-1cm^-1and 37.6%,respectively.In vitro CCK-8 assay exhibited that the cytotoxicity of AsP-BSA nanosheets was obviously declined as compaired with As₂O₃at the same As concentration.CCK-8 protocol and flow cytometric apoptosis assay confirmed that the apoptotic percentage for 4T1 cancer cells incubated with NIR laser-treated AsP-BSA nanosheets was higher than that treated with AsP-BSA nanosheets only,demonstrating that AsP-BSA nanosheets could be transformed from low toxic/nontoxic AsP crystals to highly toxic arsenic species via NIR laser treatment.The result of XPS exhibited that the area percentage of As₂O₃in As 3d XPS spectrum was obviously increased after NIR laser irradiation,achieving the in-situ transformation from low toxic/nontoxic AsP crystals to form highly toxic arsenic species.The in vivo biocompatibility assay demonstrated that AsP-BSA nanosheets caused no significant inflammation and injury in each treated mice.The in vivo data verified that these AsP-BSA nanosheets,featured with strong photothermal conversion efficiency in NIR-II biowindow and concomitantly high PA contrast,could behave as both photothermal agents and PA imaging agents for tumor theranostics.Importantly,these fabricated AsP-BSA nanosheets could in-situ be transformed from low toxic/nontoxic AsP crystals to highly toxic arsenic species by local hyperthermia,confirming the synergy between photonic therapy in NIR-II and enhanced As-based chemotherapy.Conclusion:Firstly,2D O-BiOCl-PVP nanosheets were constructed and developed for efficient cancer theranostics by a specific defect engineering strategy.After rationally tuning and optimizing the energy bandgap,near-infrared optical absorbance,and photothermal conversion performance,the designed oxygen vacancy-engineered O-BiOCl-PVP nanosheets were employed as multifunctional theranostic agents for efficient CT and PA dual-modality imaging-guided photonic cancer ablation,which has been systematically demonstrated both in vitro and in vivo.Sencondly,distinct 2D AsP-BSA nanosheets were engineered by adopting an alloy strategy,which can confine toxic arsenic into AsP crystals,thus significantly improving the biosafety and biocompatibility of arsenic-based chemotherapeutic drugs.The high light absorption and strong photothermal-conversion efficiency（37.6%）in the second near infrared biowindow of AsP nanosheets not only endowed them with desirable contrast-enhanced photoacoustic imaging properties,but also achieved efficient local tumor hyperthermia,which further synergistically triggerred the in-situ transformation from low toxic/nontoxic AsP crystals into highly toxic arsenic species,exerting a strong arsenic-mediated antineoplastic effect.Both in vitro and in vivo data verified the synergy between photonic therapy and enhanced chemotherapy as enabled by AsP-BSA nanosheets,paving the way for efficient nanomedicine-enabled arsenic-based chemotherapeutic solid tumor treatment.