Synthesis Of Hydrogen Peroxide-responsive Self-illuminating Nanoparticles And Their Applications In Imaging And Photodynamic Therapy Of Cancer

Tumors have been threatening human's health on a global scale.Increased generation of reactive oxygen species?ROS?has been long observed in cancer.In addition,accumulated evidence supports that tumor cells are more susceptible to ROS-induced cell death than normal cells.This may be due to the fact that tumor cells have relatively high levels of ROS,which approach the threshold of cytotoxicity.As the most abundant component of ROS,hydrogen peroxide?H₂O₂?is generally elevated in both tumor cells and tumor microenvironment.Therefore,abnormally overproduced H₂O₂ provides a useful biochemical cue for the design of new strategies for molecular imaging and selectively killing of malignant cancers.Due to the advantages of noninvasiveness and convenience,optical imaging techniques and photodynamic therapy have received extensive attention.However,both of them are faced with the limitation of biological tissue penetration of light and the biosafety of materials used,which hinder their applications and conversions.Based on the previous research of our group,an hydrogen peroxide-responsive nanoparticle?CLP NP?was synthesized by chemiluminescence small molecule luminol,second-generation photosensitizer chlorin e6?Ce6?and FDA-approved biocompatible polyethylene glycol?PEG?.Taking advantage of the excessive existence of H₂O₂ in tumor and chemiluminescence resonance energy transfer?CRET?,near-infrared luminescence imaging for tumors and photodynamic therapy without external light source excitation are realized through CLP NP.Methods1.Synthesis and characterization of an amphiphilic material CLPBriefly,Ce6 was activated by activator EDC/NHS.Then luminol and mPEG-NH₂ were added successively for reaction.Finally,the reaction mixture was dialyzed,filtered and lyophilized to obtain amphiphilic materials CLP.The fourier-transform infrared spectra,¹H NMR spectra,Matrix-assisted laser desorption/ionization time-of-flight mass spectra,2D and 3D absorption and emission fluorescence spectra were investigated.2.Fabrication and characterization of hydrogen peroxide-responsive CLP NPCLP NP were acquired by dissolving amphiphilic material CLP in aqueous solution.Particle size,?-potential value,morphology,the change of fluorescence intensity of different concentrations of CLP NP,¹H NMR spectra dissolved in D₂O or DMSO-d₆ were recorded.The effect of dilution on the particle size and scattered light intensity of CLP NP was investigated.The particle size changes of CLP NP in different solvents were investigated at different incubation times.3.Theoretical and experimental verification of the luminescence properties in CLP NPThe luminescence intensity of CLP NP,Ce6-PEG and luminol-Na mixed with H₂O₂were detected by BPCL-2-KGC ultraweak luminescence analyzer.The optimized geometry of the simplified molecule Ce6-Luminol was calculated by the Gaussian 09,and the linear distance between the conjugate structure of Ce6 and the benzene ring structure of luminol was calculated.The overlap between the normalized absorption spectrum of Ce6 and the normalized luminescence spectrum of luminol-Na was investigated.The luminescence adding various emission filter?620-800 nm in 20 nm steps or open?were acquired with in vivo imaging system?IVIS?after mixing CLP NP or luminol-Na with H₂O₂.4.Influence of different factors on luminescence of CLP NPFirstly,the effect of nanoparticle concentration and H₂O₂ concentration on the luminescence intensity of CLP NP was evaluated by BPCL-2-KGC ultraweak luminescence analyzer.Secondly,the advantages of luminescence imaging of CLP NP under pathological H₂O₂ concentrations?0-100?M?were investigated by IVIS.Then,the effect of antioxidants NAC or GSH and surfactant SDS on the luminescence of CLP NP was explored.5.Quantification of intracellular H₂O₂ levels in different tumor cellsVarious tumor cells were separately collected,ultrasonicated,centrifuged,and the supernatant was taken to detect H₂O₂ concentration and protein content.6.In vitro luminescence imaging of CLP NP in different cancer cellsTime-and dose-dependent cellular uptake of CLP NP in 4T1 cells were performed.Then,the effects of cell type,cell number,nanoparticle concentration,and various antioxidants on the luminescence imaging behavior of CLP NP in 4T1 or A549 cells were investigated.7.Determination of hydrogen peroxide in tumor tissuesFirstly,subcutaneous tumor models of 4T1,HCT116 and A549 were established.Secondly,fresh tumor tissues were collected,prepared into frozen slices,stained with frozen slice hydrogen peroxide detection kit.Additionally,tumor tissues were homogenized,centrifuged,and the supernatant was taken to detect H₂O₂ concentration.8.In vivo luminescence imaging of CLP NP in 4T1 subcutaneous tumor modelFirst,male BALB/c mice were subcutaneously injected with CLP NP,mice bearing4T1 tumors were intratumorally injected CLP NP or luminol-Na.The luminescence and fluorescence images were acquired by IVIS.Secondly,different tissues from tumor-bearing mice were homogenized,centrifuged,and supernatants were taken and mixed with CLP NP or luminol-Na for luminescence imaging.Finally,intratumoral injection of NAC was performed two hours before the administration of CLP NP,and the inhibition of luminescence and fluorescence of CLP NP were detected by IVIS.9.Theoretical and experimental verification of the generation of singlet oxygen in CLP NPThe energy gap between the singlet state S₁ and the triplet state T₁ of the simplified molecular Ce6-Luminol excited state was calculated by Gaussian 09.The effect of nanoparticle concentration and H₂O₂ concentration on the generation of singlet oxygen?¹O₂?by CLP NP was investigated by using SOSG.10.In vitro cytotoxicity evaluation of CLP NPAfter 4T1,HCT116 or A549 cells were incubated with CLP NP for 4 h,intracellular ROS were detected by DCFH-DA probe.MTT assay was used to detect the survival rate of4T1,HCT116 and A549 cells after incubation with different concentrations of CLP NP for24 h and calculate the IC₅₀.11.In vivo antitumor efficacy of CLP NP on A549 subcutaneous tumor after intravenous deliveryMice bearing A549 subcutaneous tumor were treated with saline,1 mg/kg CDDP,6mg/kg CDDP or CLP NP?Ce6 unit concentration at 25 mg/kg?by intravenous injection every three days,respectively.The body weight and tumor volume were monitored every 3days.After 14 days,the mice were euthanized,the tumors were taken and photographed,and blood was collected to detect hematological parameters and biochemical markers,and the organ index was calculated.12.In vivo antitumor therapy with CLP NP on A549 subcutaneous tumor after intratumoral deliveryMice bearing A549 subcutaneous tumor were treated with saline,or CLP NP?Ce6 unit concentration at 3.25 mg/kg?by intratumoral injection every three days,respectively.The body weight and tumor volume were monitored every 3 days.After 30 days,the mice were euthanized for additional analyses.13.Quantification of intracellular ROS levels in 4T1 tumors after treatment with CLP NP by intratumoral injectionMice bearing 4T1 subcutaneous tumor were treated with CLP NP?Ce6 unit concentration at 3.25 mg/kg?by intratumoral injection.After 7 h,the mice were sacrificed,and the tumor tissues were removed to prepare single-cell suspension and intracellular ROS were detected by DCFH-DA probe.14.In vivo antitumor therapy with CLP NP on 4T1 subcutaneous tumor after intratumoral deliveryMice bearing 4T1 subcutaneous tumor were treated with saline or CLP NP at 3.25mg/kg or 6.5 mg/kg of Ce6 by intratumoral injection every three days,respectively.The body weight and tumor volume were monitored every 3 days.After 17 days,the mice were euthanized for additional analyses.Lungs were soaked in a Bouin's solution or fixed and stained with H&E to observe metastatic lesions.15.Preliminary study on the mechanism of CLP NP-mediated antitumor activityAfter incubation with CLP NP,the apoptosis,target aggregation in mitochondria,mitochondrial membrane potential and apoptosis-related proteins in A549 cells were detected.16.Study on pharmacokinetic of CLP NP after intravenous injectionBALB/c mice were intravenously injected with CLP NP?Ce6 unit concentration at3.25 mg/kg?.At predetermined time periods,whole blood and major organs were collected,urine and feces were collected by metabolic cages,After relevant treatment,CLP concentrations were quantified by HPLC.17.Medium-long safety evaluation in mice of CLP NPThis experiment was divided into two phases.The first phase was Intervention period.BALB/c mice were intravenously injected with saline or different doses of CLP NP?Ce6unit concentrations at 25 mg/kg,50 mg/kg,100 mg/kg,respectively?every three days.The body weight of the mice was recorded and the state of the mice was observed every three days.On the 30th day,five mice in each group were sacrificed,and their organ index,hematological parameters,biochemical markers,organ and lymph node stained with H&E were examined.The second stage was the recovery period.Mice were fed normally without administered.On the 58th day,the remaining mice were sacrificed,and the detection indicators are the same as the first phase.Results1.The amphiphilic material CLP was successfully synthesized by chemical condensation reaction and characterized by various means.2.The amphiphilic material CLP can be self-assembled into nanoparticles in aqueous solution,with a hydrodynamic diameter of 178±3 nm and zeta-potential of-7.7±0.3 mV.A typical circular core-shell structure was observed by transmission electron microscopy?TEM?.The fluorescence intensity of CLP NP in water initially increased with its concentration,then started to decrease as its concentration increased further,showing an inverted V-shape relationship between the concentration and the fluorescence intensity.Furthermore,the proton signals of both Ce6 and luminol were notably attenuated in the ¹H NMR spectrum of CLP recorded in D₂O,which was dramatically different from that in DMSO-d₆.In addition,no significant decrease in the mean diameter was detected upon dilution of CLP NP in deionized water,although the scattering intensity was notably reduced.Comparable size distribution profiles were detected for CLP NP in water,PBS,cell culture medium and FBS.And particle size of the CLP NP was stable for 4 days in water.3.The luminescence signals of CLP NP and luminol-Na in the presence of H₂O₂ can be detected by BPCL-2-KGC ultraweak luminescence analyzer,while the Ce6-PEG conjugate?Ce6-PEG?did not show any luminescence in the absence of the CRET donor luminol.According to the calculation by using the Gaussian 09 program,the linear distance between luminol and Ce6 units in the CLP conjugate is approximate 0.6 nm,which is within the range generally required for CRET.In addition,luminescence of luminol and absorbance of Ce6 are overlapped to a certain degree.Further,luminescent signals of CLP NP remained upon incubation with H₂O₂ and adding emission filters with various wavelengths?620-800 in 20 nm steps?,showing a peak at 680 nm.4.The luminescence intensity of CLP NP showed an inverted V-shape with its concentration,and had positively correlated with the concentration of H₂O₂.In addition,CLP NP showed strong and sustained luminescence by pathologically relevant levels of H₂O₂?0-100?M?,compared to luminol-Na.Moreover,luminescence of CLP NP was notably attenuated by antioxidants NAC and GSH.Lastly,surfactant SDS could increase the luminescence intensity of CLP NP to some extent.5.Firstly,the relative levels of H₂O₂ in a series of tumor cell lines were quantified.And 4T1 cells showed the highest intracellular H₂O₂ level based on analysis of the cell number.Secondly,4T1 cells showed time-and dose-dependent cellular uptake of CLP NP as quantified by flow cytometry.In addition,after incubation with tumor cells,the luminescence intensity of CLP NP had positive correlation to intracellular H₂O₂concentration,cell number,and nanoparticle concentration.And it had inverse proportion to broad-spectrum antioxidants NAC and GSH,catalase CAT.Superoxide anion inhibitor Tempol and the hydroxyl radical inhibitor SF had little effect on the luminescence intensity.6.The levels of H₂O₂ in subcutaneous tumor were quantified which in 4T1 tumors confirmed the highest.Compared with luminol-Na,CLP NP had excellent luminescence intensity and sensitivity in the 4T1 subcutaneous tumor model and its homogenates.Moreover,CLP NP had dual luminescence-fluorescence imaging capabilities.Pretreatment of tumor models with antioxidant NAC significantly inhibited the luminescence ability of CLP NP,but had no effect on fluorescence imaging.7.Theoretical calculations showed that the energy gap between the singlet state S₁ and the triplet state T₁ of the simplified molecular Ce6-Luminol excited state was 0.855 eV,which is desirable for intersystem crossing.The generation of ¹O₂ of CLP NP was detected by using SOSG as a probe,affected by the concentration of nanoparticle and H₂O₂.Intracellular ROS levels significantly increased after incubation with CLP NP.In particular,4T1 cells showed the highest ROS level.MTT assay demonstrated that CLP NP showed a dose-dependent cytotoxicity.The IC₅₀ values on various cells were well-correlated with the relative intracellular ROS levels.8.After treatment by intravenous administration,CLP NP not only showed antitumor activity compared to CDDP,a typical chemotherapy drug,but also avoided toxic effects,as indicated by no significant changes in the body weight,organ index,and number of platelets.Intratumoral injection of CLP NP dose is 13%of intravenous administration,which still showed a significant inhibitory effect on A549 subcutaneous tumors.9.Intratumoral injection of CLP NP significantly amplified ROS levels in subcutaneous 4T1 tumors.In addition,intratumoral injection of CLP NP not only effectively suppressed primary tumor growth by inhibiting proliferation,promoting apoptosis,and reducing angiogenesis,but also remarkably prevented lung metastasis of 4T1tumors.10.Flow cytometry analysis found that the apoptosis of A549 cells increased with the dose of CLP NP.CLP NP targeted the mitochondria of A549 cells observed by confocal microscopy.In addition,CLP NP induced mitochondrial membrane depolarization in a dose-and time-dependent manner.After incubation with CLP NP in A549 calls,apoptosis-related proteins caspase-3 and caspase-9 were activated,with little change in caspase-8,investigated by western blot.11.In vivo pharmacokinetic study after intravenous injection was performed by HPLC.CLP NP cleared faster in plasma.CLP NP distributed in examined major organs were decreased,and about half of the CLP NP were excreted through the excrement in prototype form.Medium-long safety study indicates that CLP NP did not cause irreversible damage to mice.Conclusions1.Core-shell structured nanoparticle,CLP NP,with good stability was successfully synthesized.CLP NP produce near-infrared luminescence through activated Ce6 unit based on the chemiluminescence of the Luminol unit and the CRET effect under H₂O₂ oxidation.2.Based on the pathological features that H₂O₂ increase abnormally in tumor cells and tumor tissues,CLP NP can perform near-infrared luminescence imaging on tumor cell lines and subcutaneous tumor models,with good specificity,sensitivity and high luminescence intensity.3.Based on the characteristics of photosensitizer,CLP NP produce amplified-cytotoxic ¹O₂ to generate in-site photodynamic therapy of cancer without exogenous excitation.CLP NP inhibits the growth and metastasis of subcutaneous tumors by inhibiting proliferation,promoting apoptosis,and reducing angiogenesis.Further studies revealed that CLP NP triggers mitochondrial toxicity through ¹O₂,induces mitochondrial membrane depolarization,and triggers mitochondria-mediated caspase-associated endogenous apoptotic pathway in a concentration-dependent manner.4.After intravenous injection,CLP NP can be effectively removed and have good biocompatibility.In summary,we synthesized hydrogen peroxide-responsive nanoparticle CLP NP.Under the oxidation of H₂O₂,the Luminol unit in CLP NP generated chemiluminescence,and then activated the Ce6 unit through CRET effect.On the one hand,blue chemiluminescence of luminol unit converted into near-infrared luminescence with strong tissue penetrating ability for imaging;on the other hand,activated Ce6 in CLP NP synchronously produced singlet oxygen,to generate photodynamic therapy without external light source excitation.The CRET effect as well as the near-infrared luminescence and singlet oxygen generation under H₂O₂ oxidation were verified theoretically and experimentally.The content of H₂O₂ in tumor cell lines and tumor tissues was specifically examined.The near-infrared luminescence imaging and photodynamic therapy of CLP NP were examined in various tumor cell lines as well as in vivo tumor models.This responsive nanoparticle is simple to prepare and has good biocompatibility,and provides a new strategy for imaging and treatment of tumor.