Synthesis Of Bioresponsive Luminescent Nanoparticles Based On Luminol/Cyclodextrin And Their Applications In Imaging And Therapy Of Acute Liver Injury

The liver is an important organ to maintain human health.With the change of people’s lifestyle,more risk factors can lead to liver damage,and further cause a series of liver related diseases,including hepatitis,fatty liver,liver fibrosis,liver cirrhosis and liver cancer,which are seriously threatening human life and health.Therefore,the early diagnosis or treatment is critical to prevent and control liver-related diseases.Here we reported a myeloperoxidase（MPO）-responsive luminescent nanoparticle based on luminol-conjugatedα-cyclodextrin（α-CD）for the imaging and treatment of neutrophil-mediated alcohol liver injury（ALI）and acute liver failure（ALF）.The luminescence intensity of LaCD NP was intimately dependent on the level of ROS and MPO activity.In mouse models of alcohol-induced ALI and CCl₄-induced ALF,LaCD NP enabled precise quantification and tracking of the number and dynamics of neutrophils in livers.In both cases,changes in the luminescence intensity are well consistent with time-dependent profiles of neutrophils,MPO,and other parameters relevant to the development of liver injury.Meanwhile,LaCD NP can also be effective for treatment of ALI and ALF by eliminating high levels of ROS,inhibiting the migration of macrophages and neutrophils,and inhibiting the secretion of MPO,IL-8,TNF-α,IL-1βof neutrophils.Importantly,both in vitro and in vivo experiments demonstrated good safety of LaCD NP.Methods1.Synthesis and characterization of luminol-conjugatedα-cyclodextrin luminescent materials LaCDBriefly,under the protection of nitrogen,the hydroxyl group ofα-CD is firstly activated by CDI to form the intermediate CDI-α-CD,and then LaCD is formed by conjugating luminol through amide bond.Subsequently,LaCD was characterized by FT-IR,¹H NMR,¹³C NMR and MALDI-TOF-MS as well as UV-Vis.2.Preparation and characterization of LaCD nanoparticles（LaCD NP）LaCD was dissolved in DMF,distilled water was added gradually,and LaCD NP was formed by self-assembly through nanoprecipitation.LaCD NP with different particle size was prepared by changing the solvent ratio,reaction temperature or reaction time.Cy5/LaCD NP and Cy7.5/LaCD NP were prepared by the same method.Subsequently,the morphology and particle size of the LaCD nanoparticles were characterized.3.Stability and hydrolysis of LaCD NP in vitroLaCD NP was dissolved in distilled water or serum medium,then its stability was tested.The hydrolysis was detected in different concentrations of H₂O₂ and NaClO solutions or in solutions with varied pH values,and the hydrolysis products were characterized by ¹H NMR and MALDI-TOF-MS.4.The luminescent properties of LaCD NP in vitroUnder different ROS environment,the luminescence properties of LaCD NP in vitro was measured by a Optical fiber spectrometer and a BPCL Ultra-Weak Luminescence Analyzer as well as an IVIS Spectrum imaging system.5.Cellular uptake of LaCD NPBalb/c female mice were intraperitoneally injected with 1 mL of 3%thioglycolate solution,and the peritoneal neutrophils was extracted after 4 h.After Cy5/LaCD NP was incubated with neutrophils for different times,the phagocytic behavior was observed by confocal laser microscopy and detected by flow cytometry.Through similar procedures,the dose-dependent internalization profile was examined.6.Imaging of neutrophils by LaCD NPNeutrophils were first stimulated with 100 ng PMA,then DCFH-DA was added,ROS were observed by confocal laser scanning microscopy,and MPO content was determined by ELISA.Neutrophils in black 96-well plates were stimulated by PMA,then LaCD NP was added under different conditions,luminescence signals were examined by an IVIS imaging system.7.Imaging of mice with alcoholic liver injury by LaCD NPA mouse model of alcoholic liver injury was established by oral administration of300μL 50%ethanol in C57BL/6 male mice.In order to investigate that LaCD NP targeted damaged liver,mice with alcoholic liver injury were given Cy7.5/LaCD NP through the tail vein,normal mice were injected with PBS as the control.After 10 min,the liver was removed and imaged by an IVIS imaging system.To investigate the in vivo imaging ability of LaCD NP,LaCD NP was administered by intravenous（i.v.）injection of mice with alcoholic liver injury,normal mice were injected with PBS as control.Then,in vivo luminescence imaging was performed by the IVIS Spectrum system after 5 min.Subsequently,the liver was removed and imaged.In a separate study,LaCD NP was administered i.v.injection of mice at different time after alcohol intragastric（i.g.）administration,and in vivo luminescence imaging was performed by the IVIS Spectrum system after 5 min.8.Imaging of mice with acute liver failure by LaCD NPA mouse model of acute liver failure was established by intraperitoneally（i.p.）injected with 100μL 25%CCl₄ in C57BL/6 male mice.In order to investigate that LaCD NP targeted damaged liver,mice with acute liver failure were given Cy7.5/LaCD NP through the tail vein,normal mice were injected with PBS as the control.After 10 min,the liver was removed and imaged by an IVIS imaging system.To investigate the in vivo imaging ability of LaCD NP,LaCD NP was administered by i.v.injection of mice with acute liver failure,normal mice were injected with PBS as control.Then,in vivo luminescence imaging was performed by the IVIS Spectrum system after 5 min.Subsequently,the liver was removed and imaged.In a separate study,LaCD NP was administered by i.v.injection of mice at different time after CCl₄ stimulation,and in vivo luminescence imaging was performed by the IVIS Spectrum system after 5 min.9.Treatment of mice with acute liver injury by LaCD NPLaCD NP was administered by i.v.injection of C57BL/6 male mice after oral administration of 50%ethanol for 1 h,the levels of serum liver function and liver neutrophils,MPO and H₂O₂,as well as cytokines including TNF-αand IL-1βwere detected after 11 h.Similarly,LaCD NP was administered by i.v.injection of C57BL/6 male mice after intraperitoneally injected with 25%CCl₄ for 1 h,the levels of serum liver function and liver neutrophils,MPO and H₂O₂,as well as cytokines including TNF-αand IL-1βwere detected after 23 h.10.Preliminary study on the therapeutic mechanism of LaCD NPAfter LaCD NP was incubated with H₂O₂ and NaClO at different concentrations for48 h,their consumption was determined.After LaCD NP was incubated with macrophages and neutrophils,cell migration was performed by Transwell assay.After co-incubation with macrophages at different concentrations of LaCD NP under LPS stimulation for 1 h,cytokines including TNF-α,IL-1β,MCP-1 and IL-6 were detected.Similarly,after LaCD NP was co-incubated with with neutrophils at different concentrations of LaCD NP under PMA stimulation for 1 h,cytokines including MPO,IL-8,TNF-α,IL-1β,MCP-1 and IL-6 were detected.11.Safety evaluations of LaCD NPAfter different concentrations of LaCD NP were incubated with RAW264.7macrophages,human umbilical vein endothelial cells and HepG2 human liver cancer cells for 12 h,then MTT analysis was examined.The hemolysis behavior was examined using fresh erythrocytes isolated from Sprague Dawley rats with incubaion with LaCD NP of different concentrations for 2 h.LaCD NP was injected into the tail vein of Balb/c female mice at 500 mg/kg and1000 mg/kg respectively.After 15 days,the organ indexes were calculated,hematological parameters,liver and kidney functions were detected,and the main organs were stained with H&E.Results1.LaCD was synthesized based on luminol-conjugatedα-CD,eachα-CD was linked with about 3 imidazole groups and 1 luminol unit.LaCD NP prepared by nanoprecipitation were spherical and uniform.LaCD NP shows good stability in distilled water or serum,and is hydrolyzed intoα-CD and soluble small molecules under high ROS or alkaline environment.2.In vitro luminescence performance test showed that LaCD NP has excellent luminescence performance with high luminescence intensity and long luminescence time.MPO and ClO^-can significantly increase the luminescence intensity,and 4-ABAH and Tempol can effectively inhibit the luminescence of LaCD NP.3.The extracted peritoneal neutrophils were horseshoe-like polymorphonuclear structures with a purity of about 90%.Peritoneal neutrophils can effectively phagocyte LaCD NP,and their MPO and ROS levels were significantly increased afte r PMA stimulation.Compared with luminol,LaCD NP showed significant luminescent signal in neutrophils with a longer time.4-ABAH and Tempol also effectively inhibited luminescence of LaCD NP in neutrophils.4.After oral administration with 50%ethanol,a mouse model of alcoholic liver injury was successfully replicated.Cy7.5/LaCD NP produced significant fluorescence in the damaged liver.The liver of the model mice produced significant luminescence signals after i.v.injection of LaCD NP.At different time points after alcohol intragastric administration,the intensity of luminescence was the highest at 12 h.5.After intraperitoneally injected with 25%CCl₄,a mouse model of acute liver failure was successfully replicated.Cy7.5/LaCD NP produced significant fluorescence in the damaged liver.The liver of the model mice produced significant luminescence signals after i.v.injection of LaCD NP.At different time points after CCl₄ stimulation,the intensity of luminescence was the highest at 24 h.6.LaCD NP has been shown to be effective in the treatment of alcoholic liver injury and acute liver failure.Serum ALT and AST levels were decreased,liver neutrophil infiltration was decreased,liver MPO,H₂O₂,TNF-αor IL-1βwere decreased.7.Preliminary studies in vitro confirmed that LaCD NP can eliminate high levels of ROS,inhibiting the migration of macrophages and neutrophils,and inhibiting the secretion of MPO,IL-8,TNF-α,IL-1βof neutrophils.8.In vivo and in vitro safety experiments showed that cell activity will not be affected by LaCD NP,and no obvious hemolysis phenomenon occurred.The body weight of mice,organ index,liver and kidney function have no changes after i.v.injection of 500mg/kg and 1000 mg/kg LaCD NP.Conclusions1.A amphiphilic MPO-responsive luminescent material LaCD was synthesized based on luminol-conjugatedα-CD.And the LaCD NP with regular morphology and uniform size was successfully prepared by nanoprecipitation method.LaCD NP showed good stability under normal physiological conditions,in the high ROS inflammatory environment,LaCD NP structure was damaged and hydrolyzed intoα-CD and soluble small molecules,which can be eliminated by the kidney.2.LaCD NP showed excellent in vitro luminescence performance,with high luminescence intensity and long luminescence time,which was responsive to MPO and ROS.LaCD NP can be effectively used for imaging active neutrophils for a long period of time,which dependened on MPO and ROS.3.LaCD NP can be used as a new luminescent probe to detect mice with alcoholic liver injury and acute liver failure by imaging infiltrating neutrophils,and real-time monitor the occurrence and development of liver injury.At the same time,LaCD NP can also effectively alleviate the process of acute liver injury.4.LaCD NP shows good biosafety and has no obvious side effects on the body.In summary,our newly developed nanoprobe LaCD NP can serve as an efficient,biodegradable,and biocompatible luminescent nanoprobe for in vivo dynamic detection of the initiation,progression,and resolution of neutrophil-mediated ALI and ALF.Furthermore,LaCD NP has been shown to be effective in the treatment of ALI and ALF.It is also promising for diagnosis and treatment of other liver diseases associated with neutrophils in the future.