Synthesis And In Vivo Imaging Of Multi-functional Mesoporous Silica Nanoprobe With Near-infrared And Magnetic Resonance

BackgroundGliomas are the most prevalent primary tumors in central nevers system (CNS). Eighty percent of the gliomas are malignant tumors and essentially incurable, which including anaplastic gliomas (World Health Organization (WHO) grade III) and glioblastomas (WHO grade IV). Despite aggressive multimodal therapy, including maximal surgical resection, combined radiation and chemotherapy, and adjuvant anti-angiogenic therapy, the prognosis of glioblastoma remains dismal. Recurrency and progression has been reported in some patients. Maligant gliomas tend to infiltrate into normal tissue by growing along with nerve fibers and capillaries, it is not an easy task to distinguish tumor with brain. The hyperplastic glias surrounding the lesions make it even worse to identify the border of GBM and recurrenct tumor clearly. It is critical important to detect GBM noninvasively when the tumor remain small, as well as identify tumor cells in normal neurons and the hyperplastic glias. The most effective tools for glioma detection are Magnetic Resonance Imaging (MRI) and Positron Emission Tomography (PET). However, due the poor spatial resolution and other limitions, it is not easy to identify very small gliomas from MR and PET images. Because they may mimick benign lesions which seldem disrupt the blood-brain-barrier (BBB). Tremendous efforts have been engaged in improvements of tumor detection and delineate tumor boundary noninvasively.With progression of technology and bioscience, a new discipline which was defined as molecular imaging (MI), aims to monitor biochemistry progress in vivo at cellular level emerged. MI is expected to play an important role in detect disese at the early stage, because it will allow sensitive and specific monitoring of key molecular targets and host responses associated with early events in carcinogenesis. Molecular probe is the most important and essential issue in MI. With molecular probe administration, cells and molecule can be visualized by using conventional imaging modality. As the most powerful and effective imaging tools in bioscience, MI modalities has been used prevalencely. Amounts of research has been conducted in develop more sensitive and specific probes.With the improvement of bioscience knowledge, more precious details is needed to characterize disease, which can not be illustrated by signal modality images. The combination of2or more imaging modalities to identify tumors in vivo is widely used. As the result, multi-modality image probe development is needed. Among the traditional medical imaging modalities, MRI and optical imaging have been widely used as they take the adbantage of noninvasive, free X-ray radition, and highly reproducibility. The combination of MR and optical imaging may provide high sensitive detection of tumor with more detailed anatomy structures, which will ensure more accurate tumor detection at its early stage.In this article, we focus on the precious glioma detection at the early stage by using the dual-modality image probe synthesized with mesoporous silica nanoparticle, near-infrared fluorescent heptamethine indocyanine dye IR-808, and Gd-DTPA. In vivo near-infrared fluorescent and MR imaging was performed on human glioblastoma U87-MG xenograft nude mice model.Purpose1、To evaluate the ability of near-infrared fluorescent heptamethine indocyanine dye IR-808preferential glioma and breast cancer accumulation as a non-specific optical imaging probe.2、Synthesis mesoporous silica nanoparticls with excellent biocompatibility, tunable structure, which can be used as probe of theranostic.3、Synthesis dual-modality image probe with mesoporous silica nanoparticls, near-infrared fluorescent heptamethine indocyanine dye IR-808, and Gd-DTPA. To evaluate the biocompatibility of the probe, as well as the effect of preferential accumulation in C6and U87-MG cells.4、In vivo near-infrared fluorescent and MR imaging with the dual-modality probe synthesized.Material, Methods and Results一、NIR heptamethine cyanine dye IR-808mediated glioma and breast cancer imaging1、Cell lines and cell cultureC6cells were cultured in F12K media with10%FBS,2.5%horse serum. Human embryonic kidney cells HEK293T, Human glioblastoma U87-MG and human breast cancer MDA-MB-231cells were cultured in DMEM media with10%FBS. All the cells were added with1%penicillin/streptomycin and incubated in the incubated at37℃with5%CO2. At80%confluence, cells were split1:3and cultured for one passage.2、Cell uptake study using NIR heptamethine cyanine dye IR-808 Cells (1×104per well) were seeded on vitronectincoated24-well chamber slides and incubated with medium containing5%fetal bovine serum for24hours. After the cells had attached to the chamber slides, the cells were washed with PBS and exposed to the cyanine dye at a concentration of20μmol/L in the medium. The slides were incubated at37℃and washed twice with PBS to remove excess dyes, and cells were fixed with10%formaldehyde at4℃. The slides were then washed twice with PBS and covered with glass coverslips. Images were recorded by microscopy using a778-nm excitation laser and670to810nm long pass filter or a fluorescence microscope equipped. The dye was found not to accumulate in HEK293T cells, while was found to be taken up by C6, U87-MG and MDA-MB-231cells.2、Acute toxicity studyTo determine the acute toxicity of IR-808in cells, human embryonic kidney cells HEK293T was used. Cells (1×105per well) were seeded on vitronectincoated96-well chamber slides and incubated with medium containing5%fetal bovine serum for24hours. Cells was exposed to the cyanine dye IR-808for24or48h。The acute toxicity was determined by MTT assay. The results suggested the IR-808has low toxicity effect at a very high concentration.3、Uptake and accumulation of cyanine dyes IR-808in tumors in live miceC6and MDA-MB-231cells were implanted (1×106) either orthotopically, or subcutaeous into4-to6-week-old athymic nude mice. When tumor sizes reached between0.5mm in diameter, mice were injected i.v. with IR-808at a dose of0.375mg/kg or10nmol/20g mouse body weights. Whole body optical imaging was taken at24hours. Successive observations revealed that after the initial systemic distribution and clearance, intense signals were clearly associated with the tumors implanted at various anatomic sites, with no background interfering fluorescence from the mice.二、Synthesis of dual-modality imaging probe1、Synthesis and Characterization of MSNs-NH2Ethyl acetate, ammonium hydroxide, and a mixture of TEOSand3-aminopropyl triethoxysilanewere added into an aqueous solution of hexadecyltrimethylammonium bromide and stirred for30minutes. Additional water was then added into the reaction before leaving the solution overnight under stirring.. The volume ratio of all compounds in milliliters was1CTAB (aq):0.045TEOS:0.055APTES:0.54NH4OH:0.176EtOAc:27.38H2O. The resulting solution was slightly translucent. After24hours, the reaction solution was neutralized using hydrochloric acid solution. The sample was cleaned by centrifugation and redispersed in ethanol. CTAB was remove by additional HCl。Transmission electron microscopy (TEM) images were obtained with a200-CX microscope operated at an acceleration voltage of120kV. Average particle sizes (120nm) were obtained by averaging over approximately100particles. Nitrogen physisorption isotherms of dried samples were obtained with a V-Sorb2800P physisorption instrument. The particles exhibited nitrogen sorption isotherms of type IV according to BDDT classification. Surface areas (-473m2/g) were determined according to the Brunauer-Emmett-Teller (BET) method. Calculation of the pore size (2.5nm) distributions from the adsorption branches of the isotherms was performed according to the Barrett-Joyner-Halenda5(BJH) method. Fourier Transform Infrared (FTIR) spectra were measured with NEXUS870. Elemental Analyses were performed on KBr (blank) pellet and sample pellets containing lwt%samples in KBr. All elemental analyses were conducted by Vario MICRO. 2、Synthesis and Characterization of MSNs-Gd/NIR0.7mg IR-808and4ml0.1M Gd-DTPA were added into20ml MES buffer (0.1M); after0.4mg NHS and0.6mg EDC were added, stirring at room tempicture for4h。 And then1ml MSNs-NH2was added。Fourier Transform Infrared (FTIR) spectra were measured with NEXUS870. Gd concerntration (0.009M) was determined by ICP with OPTIMA5300DV inductively coupled plasma atomic emission spectroscopy. MRexamination was performed with SIMENS3.0T MR scanner:TR=15.0ms, TE=1.6ms, matrix=512×512, Slice Thickness:2.0mm, FOV=200mm, Flip Angle=5,26. We found that r1of MSNs-Gd/NIR is much high than Gd-DTPA.3^Acute toxicity studyTo determine the acute toxicity of MSNs-Gd/NIR in cells, human embryonic kidney cells HEK293T was used. Cells (1×105per well) were seeded on vitronectincoated96-well chamber slides and incubated with medium containing5%fetal bovine serum for48hours. Cells was exposed to the cyanine dye MSNs-Gd/NIR for48h。 The acute toxicity was determined by MTT assay. The results suggested the MSNs-Gd/NIR have no obvious toxicity effect on HEK293T cells with a concentraton of20μmol/L.三、MSNs-Gd/NIR mediated glioma and breast cancer imaging1、Cell uptake study using Gd/NIR-MSNU87-MG and C6Cells (1×104per well) were seeded on vitronectincoated24-well chamber slides and incubated with medium containing5%fetal bovine serum for24hours. After the cells had attached to the chamber slides, the cells were washed with PBS and exposed to the Gd/NIR-MSN at a concentration of100mg/ml in the medium. The slides were incubated at37℃for2-3h and washed twice with PBS to remove excess dyes, and cells were fixed with10%formaldehyde at4℃. The slides were then washed twice with PBS and covered with glass coverslips. Images were recorded by microscopy using a778nm excitation laser and670to810nm long pass filter or a fluorescence microscope equipped. Near-infrared signal was foundin C6, U87-MG, which suggested the nanoprobe was uptake by cells.2、Uptake and accumulation of MSNs-Gd/NIR in tumors in live miceU87-MG cells were implanted (1×106) subcutaeous into4-to6-week-old athymic nude mice. When tumor sizes reached between0.5mm in diameter, mice were injected intratumoral with MSNs-Gd/NIR at a dose of50μl per mouse. Whole body optical imaging was taken at0、4、8、24and48hours. Successive observations revealed at different time points that after the initial systemic distribution and clearance, intense signals were clearly associated with the tumors implanted at various anatomic sites, with no background interfering fluorescence from the mice.MRI examinations were performed with MAGNETOM Trio3.0TMR scanner. The imaging parameter as following:T1WI, TR=670ms, TE=11ms, Average=4, Slice Thickness:2.0mm, FOV=60mm, Flip Angle=150; T2WI, TR=3100ms, TE=98ms, Average=4, Slice Thickness:2.0mm, FOV=60mm, Flip Angle=120; TIMap:TR=15.0ms, TE=1.73ms, Average=2, Slice Thickness:2.0mm, FOV=113mm, Flip Angle=5,26。The results illustrated that at48h intratumor near-infrared signal can be observed, while there noly slightly hyper-signal intensity on MR T1image. Nanoprobe tissue distribution studie exhibited that MSNs-Gd/NIR mainly accumulated in tumor site. Conclusions1、IR-808can is a safe agent which has the potential of preferential accumulation in gliomas.2、The mesoporous silica nanoparticls synthesized was biocompatibd and can be used as theranostic probe。3、The dual-modality image probe synthesized with mesoporous silica nanoparticls, near-infrared fluorescent heptamethine indocyanine dye IR-808, and Gd-DTPA has the ability of preferential accumulation in gliomas, and can be used in vivo near-infrared fluorescent and MR imaging probe.Innovation points1、NIR heptamethine cyanine dye IR-808has been reported as an excellent NIR probe, which can be used to visualize many malignant tumors arise form epithelial tissue, such as lung cancer and cervical cancer. However, the ability of IR-808to detect glioma has not been fully investigated. In this experiment, we try to detect orthotopic glioma with IR-808in a BABL/c nude mice model. We found that the NIR signal of IR-808can penetrate the BABL/c nude mice cranium, and ensure glioma noninvasively detection with NIR image.2、Although dual-modality image probe synthesised with organic dye, Gd-DTPA has been reported, the methods is very complicated. In this article, we first synthesized mesoporous silica nanoparticle with abundant-NH2, which can be used to connect IR-808and Gd-DTPA by condensation reaction. The dual-modality porbe MSNs-Gd/NIR illustrated higher T1relaxivity than Gd-DTPA, stable NIR quantum output. Additional, mesoporous silica nanoparticle takes the advantages of consisting of hundreds of empty channels, and hold great promise in drug delivery. MSNs-Gd/NIR may be used as multi-functional theranostic agents.