| Objective:The NIR-? fluorescence imaging(1000-1700 nm)can provide an even deeper tissue penetration(?10 mm),higher signal-to-noise ratio and better temporal-spatial resolution.Photoacoustic imaging is an emerging non-invasive method,which combines the advantages of traditional optical imaging and ultrasound imaging.The combination of NIR-? FI and PAI provides a new strategy for the development of multimodal probes for clinical disease imaging,targeted therapy,efficacy evaluation,and prognosis monitoring.Melanin is an endogenous pigment widely existing in humans.It possesses natural biocompatibility and biodegradability,easily modified chemical structure,photoprotection,metal ion chelation,and anti-oxidant properties,which has resulted in wide applications in the field of biomedical researches,including bioimaging,photothermal therapy,biosensor,theranostics,antibacterial,and tissue engineering.The purpose of this research is to build a multi-functional nanoagent with the ability of the second near-infrared fluorescence and photoacoustic dual-modal imaging and photothermal treatment based on NIR-? small molecular fluorescence dye labeled bioinspired melanin nanoparticles(MNPH2).Firstly,we study the basic characterization and performance of MNPH2,and then explore its applications as a dual-modality contrast agent and theranostics nanoagent for stem cells tracking and cancer theranostics.Methods:1.Facile Synthesis,basic characterization and performance of multifunctional nanoagent MNPH2 based on NIR-? small molecular fluorescence dye H2 labeled melanin nanoparticles.1.1 Multi-functional nanoagent MNPH2 was obtained through the chemical reaction between bioinspired MNPs and NIR-? fluorescent small molecule dye H2.1.2 The TEM,FT-IR,stability,DLS,zeta potential and UV-Vis-NIR spectrum of MNPH2.1.3 The NIR-? FI,PAI and photothermal conversion performance of MNPH2 in vitro.1.4 Study on the biosafety of MNPH2 through hemolysis experiment and high-dose injection.2.Long-term tracking hUMSCs and visible h UMSC-based regeneration for acute liver injury within NIR-? fluorescence and PA dual-modal imaging.2.1 CLSM,NIR-? fluorescence microscope and flow cytometry evaluated the uptake capacity of hUMSCs to MNPH2.2.2 The CCK-8,osteogenic and adipogenic differentiation assays analyzed the influence on proliferation viability and differentiation capacity of MNPH2 on hUMSCs.2.3 NIR-? fluorescence and PA imaging of MNPH2 labeled hUMSCs in vitro.2.4 Long-term dual-modal NIR-? fluorescence and PA imaging of engrafted hUMSCs in vivo.2.5 Dual-modal imaging monitored the transplantation and regeneration process of hUMSCs for ALF.3.NIR-? fluorescence and PA imaging-guided photothermal therapy of MNPH2 for laryngeal cancer.3.1 The influence of MNPH2 on proliferation viability,uptake capacity and photothermal toxicity were respectively evaluated by CCK-8,CLSM,NIR-? fluorescence microscope and Calcein-AM/PI.3.2 NIR-? fluorescence and PA dual-modal imaging of laryngeal cancer in vivo.3.3 The evaluation of therapeutic effect based on dual-modal imaging-guided photothermal therapy in vivo.Results:1.The multi-functional nanoagent MNPH2 with good biocompatibility,NIR-? FI,PAI,and photothermal conversion performance was facilely synthesized via a one-step method.1.1 The nanoagent MNPH2 was synthesized through the EDC/NHS reaction based on activated carboxyls of H2 and terminal amines of MNP-PEG,which formed an amide bond after dehydration condensation.And there were 22 H2 molecules per nanoparticle.1.2 MNPH2 possessed good water solubility and physiological stability.The TEM image of MNPH2 revealed a uniform size and good dispersity with a round shape.The FT-IR spectra revealed the weak absorption peaks at about 1690 cm-1 represented amide I,proving the success of the EDC/NHS reaction.The DLS result showed the average hydrodynamic diameter of MNPH2 was nearly 33 nm.The zeta potential was about-14m V.The UV-Vis-NIR spectra showed MNPH2 had a characteristic absorption peak in the NIR and the absorption peak was 768 nm.1.3 MNPH2 exhibited good NIR-? FI and PAI performance.The NIR-? fluorescence and PA signal intensities were depended greatly on the concentrations of MNPH2,which showed a good linear relationship and an excellent correlation coefficient according to the changes in the concentrations.The MNPH2 had high photothermal conversion efficiency and good photothermal stability.1.4 Hemolysis experiment indicated MNPH2 did not disrupt erythrocytes or cause hemolysis.The mice did not occur acute liver or kidney toxicity and apparent pathological changes in the major organs after high-dose injection with MNPH2.2.Long-term tracking hUMSCs and visualization of the hUMSCs treatment process of acute liver injury based on NIR-? fluorescence and PA dual-modal imaging nanoagent MNPH2.2.1 The images of CLSM and NIR-? fluorescence microscopy confirmed that the MNPH2 was successfully taken up by the hUMSCs through a simple co-incubation method.The quantitative analysis of FCM showed that the cellular uptake efficiency reached up to 98.57%after 4 h incubation.2.2.The CCK-8 assay confirmed concentrations within a range from 0 to 800?g/m L of MNPH2 tended to have an unnoticeable influence on cell viability.The cell staining of adipogenic and osteogenic differentiation potentials indicated MNPH2 had no effect on the differentiation capacity of hUMSCs.2.3 MNPH2 labeled hUMSCs exhibited bright NIR-? fluorescence and PA signals, and the signal intensity enhanced with the increasing number of hUMSCs.2.4 The subcutaneous transplantation site of MNPH2 labeled hUMSCs emitted NIR-? fluorescence and PA signals,which gradually weaken over time and disappeared on the21st day.2.5 Both NIR-? FI and PAI confirmed that most of hUMSCs were settled in the injured liver tissue after transplantation and the number of hUMSCs reached the maximum after 6 h.NIR-? FI also proved that some hUMSCs can be excreted by the gut-liver axis.The body weight,hematological and histopathological measurements demonstrated MNPH2 labeled hUMSCs played a significant protective role in the prevention of ALI and is absolutely not a transient effect.3.NIR-? FI and PAI guided PTT for laryngeal cancer based on MNPH2.3.1 The result of CCK-8 assay showed that MNPH2 had no obvious influence on cell viability.While MNPH2 will release heat to kill Hep-2 cells once laser radiation,resulting in a significant decrease on cell viability.NIR-? fluorescence microscope and CLSM showed that Hep-2 also took up MNPH2 after co-incubation.The Calcein-AM/PI live and dead cell staining once again confirmed that the heat generated by MNPH2 is sufficient to kill Hep-2 cells.3.2 NIR-? FI and PAI of laryngeal cancer in vivo showed that the tumor was gradually‘lighted up'after MNPH2 injection.The signal of tumor site reached a maximum at 8 h and then weakened.The NIR-? FI images of the major organs and tumor tissue indicated that MNPH2 is mainly excreted through liver and intestine metabolism.3.3 After NIR-? FI/PAI guided irradiation,the local temperature of laryngeal cancer increased to 59.2?,which succeeded in killing cancer cells.The body weight,tumor volume and HE staining proved MNPH2 had a great PTT effect without side effects.Conclusion:In this study,we synthesized a bioinspired multi-functional nanoagent MNPH2 that integrated NIR-? FI,PAI and PTT through a one-step method.The nanoagent probe has the following advantages:1)good water solubility and physiological stability;2)excellent NIR absorption;3)good biosafety;4)NIR-? FI and PAI performance;5)high photothermal conversion efficiency and photothermal stability.The nanoagent realized to long-term track hUMSCs and visualize the hUMSCs treatment process of acute liver injury in vivo.Meantime,the nanoprobe could also non-invasively monitor tumors in real-time and conduct NIR-? FI and PA dual-modality imaging guided photothermal treatment of laryngeal cancer.In short,MNPH2 can be utilized in stem cell tracking and regenerative medicine research,as well as cancer theranostics,which promoted the exploration of multifunctional nanoprobes based on melanin in the field of biomedicine and further prepared for its potential clinical transformation. |
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