PH-sensitive Manganese-loaded Carbonaceous Danospheres: A Theranostic Agent For Magnetic Resonance Imaging-guided Photothermal Therapy

BackgroundOver the past decades,cancer has been a major public health problem worldwide.The vital influence on the cancer progression and prognosis between initial symptoms and cancer diagnosis is difficult to be estimated.In recent years,with the development of nanotechnology,multifunctional nanomaterials integrating biomedical imaging and treatment with optimized therapeutic effect and monitoring the therapeutic response have attracted a great deal of attention.In the medical field of oncology,important value and potential have been revealed.Up till now,there have been a large number of imaging modalities applied in nanomedicine to improve the diagnostic rate,such as magnetic resonance imaging?MRI?,photoacoustic imaging?PA?,X-ray computed tomography?CT?and so on.Among different clinical imaging modalities,magnetic resonance imaging?MRI?is one of the most widely used imaging and diagnostic techniques.MRI offers several advantages over other clinical modalities,including high spatial resolution,non-invasiveness,high anatomical contrast,outstanding capacity of differentiating soft tissues and lack of ionizing radiation.Therefore,MRI-based theranostics has received wide attention and is now becoming one of the hot spots in cancer theranostics.At present,Gd-containing contrast agents?CAs?widely used in clinic are thought to be associated with renal systemic fibrosis?NSF?of patients with impaired renal function,hypersensitivity and renal fibrosis skin disease?NFD?.Five unpaired electrons outside Mn nucleus are considered to be the most promising T1-weighted CAs that can replace Gd CAs due to their long relaxation time.One of the vital obstacle in cancer therapy is to explore tumor-specific strategy that do not damage normal tissues.In recent years,photothermal therapy?PTT?utilizing near infrared?NIR?light-absorbing agents to generate heat and "boil" tumor cells without any harm to normal cells and tissues has attracted increasing attention for its minimal invasiveness and potential effectiveness.Unlike cancer traditional therpay?surgery,chemotherapy and radiotherapy?,PTT performs two levels of selectivity:passive/active-targeting to tumor of photothermal agents and NIR light beams focused directly on the tumor sites.Therefore,the combination of MRI and PTT of nanoparticle will open up new prospects for the theranostics of cancer.Based on thesis above,carbonaceous nanospheres?CNSs?were prepared from glucose by dehydration and aromatization.Mn2+ were then bonded to its surface through redox reaction between CNSs and MnCl₂ to obtain manganese-loaded carbonaceous nanospheres?Mn-CNSs?.The manganese-loaded carbonaceous nanopheres?Mn-CNSs?synthetized by our groups have several superiorities:1,Mn-CNSs can be used as near infrared light absorber for photothermal therapy;2,manganese-based contrast agents have been extensively studied as MR T1 contrast agents;3,the pH responsiveness of Mn-CNSs makes it easier to release Mn2+ under acidic conditions with a pH-dependent imaging effect.PurposeTo investigate the in vivo and in vitro MR imaging,photothermal therapy,biodistribution and blood circulation of pH-sensitive manganese-loaded carbonaceous nanospheres?Mn-CNSs?realizes the integration of theranostics of cancer.MehtodsCNSs were synthesized according to previous reports.Briefly,anhydrous glucose?4 g?was dissolved in water?35 mL?.Then the homogeneous solution was transformed into a Teflon-lined autoclave to be heated and kept at 180 ? for 6 h.Finally,the obtained brown products were separated by centrifugation?12000 rpm,10 min?and the precipitate was washed with ethanol and water.The resulting product was dried for further use.A certain amount of dried CNSs were dispersed in 50 mL water under ultrasonication and MnCl₂·4H2O?1 g?was added.Then the final solution was ultrasonicated for another 0.5 h and stirred for 4 h at room temperature.Finally,the obtained Mn-CNSs were washed with ethanol and water.The morphological characteristics and elemental composition of Mn-CNSs nanoparticles were observed by transmission electron microscopy?TEM?,scanning electron microscopy?SEM?and elemental analysis.The GE Signa HDX 3.0 T MR scanner measures the MR relaxation rate?r1?of Mn-CNSs at different pH.The toxicity of Mn-CNSs to cells was verified by CCK8 assay.Chronic toxicity of Mn-CNSs to healthy Balb/c mice at 6-8 weeks was evaluated by H&E staining.TEM and inductively coupled plasma mass spectrometry?ICP-MS?were used to investigate cellular uptake of Mn-CNSs.Near infrared laser?NIR?was employed to irradiate the tumor cells,and the treatment effect of Mn-CNSs were detect by Hoechst/PI double staining and flow cytometry analysis.Female 4T1 tumor-bearing Balb/c mice were injected with tail vein?4 mg Mn/Kg?for MR imaging and irradiated with 2 W/cm2 808nm near-infrared laser for photothermal therapy.Results1.The scanning electron microscopy?SEM?and transmission electron microscopy?TEM?images displayed the uniform spherical structure with smooth surface.Energy dispersive X-ray?EDX?mapping images were used confirm the elemental composition?manganese?Mn?,chlorine?Cl?and carbon?C??of Mn-CNSs.2.In vitro the results of MRI T1 relaxivity test showed r1 value in the acidic environment with an increase by 5-fold?from 5.42 mM-1s-1 to 0.18 mM-1s-1?,due to excellent pH responsiveness of Mn-CNSs.3.CCK-8 assay showed that cells viabilities were approximately 90%-110%even when the cells were cultured with Mn@CNSs at the concentration as high as 200?g/mL for 48 h.From H&E images,there were no observable tissue damage and inflammatory lesions in major organs compared with control group,suggesting low organs/tissues toxicity of Mn-CNSs.4.The results of TEM and ICP-MS showed that the pro-uptake and post-uptake content of Mn2+ in MCF-7,macrophages and MCF-10A cells were 11.66 ± 2.00 and 40.08 ± 2.90,5.44 ± 1.53 and 18.99 ± 2.07,1.24 ± 0.58 and 4.83 ± 4.83 fg per cell,respectively5.In vitro photothermal therapy:Hochest/PI double staining and flow cytometry analysis revealed that apoptosis and necrosis rate of MCF-7?21.69%?were higher than the control group?5.49%?after photothermal therapy?P<0.05?.In vivo photothermal therapy:the temperature of tumor increased 29 ? in intravenous injection group,intratumoral injection group increased 32 ?.The necrosis of photothermal treatment groups was more serious than that of the control group from H&E staining of tumor after photothermal therapy.6.In vivo MR imaging after intravenous injection showed that the tumor tissue was strengthened by nearly 1.5 times and liver tissue enhancement was not obvious.ConclusionManganese-loaded carbonaceous nanospheres?Mn-CNSs?can be used as a fine biocompatible and pH-sensitive contrast for combining targeted MRI with accurate photothermal therapy of 4T1 tumor-bearing mice,showing important value and potential in the integration of theranostics of cancer.