基于介孔二氧化硅双模态多功能探针的制备及实验研究
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摘要
研究背景和意义
胶质瘤是中枢神经系统最常见的原发性肿瘤,其发病率高,预后差。胶质瘤中约80%的病变为恶性,主要包括胶质母细胞瘤和间变性胶质瘤。虽然近年来出现了以手术为主,联合放疗、化疗的综合治疗方案,和一些针对肿瘤血管新生的靶向治疗药物,但恶性胶质瘤患者的5年生存率仍然没有得到显著改善。部分胶质瘤患者在接受治疗后还出现肿瘤复发进展、恶性程度增高的现象。这主要是因为恶性胶质瘤特别是胶质母细胞瘤呈浸润性生长,肿瘤细胞沿神经纤维和毛细血管侵犯生长,与正常脑组织、神经纤维和血管分界不清,手术难以彻底切除。此外,手术和放疗区域大量增生的神经胶质,也给残留和复发肿瘤组织的检测带来了很大困难。因此对胶质瘤进行非侵入性的早期诊断、准确定位和边界显示,并区分正常脑组织、肿瘤和增生的神经胶质,对治疗和预后具有十分重要的意义。目前胶质瘤的诊断主要依靠影像学手段,特别是磁共振和核素扫描。但磁共振和核素检查对体积较小、血脑屏障破坏较轻的肿瘤检测的敏感度和特异度较低。如何在提供详细解剖学细节的基础上实现对胶质瘤高敏感度、高特异度的非侵入性的显像,成为全世界范围内研究人员和医务工作者面临的巨大挑战。
随着科技的进步和生命科学的发展,在最近几十年出现了以能够在活体状态下利用影像学方法在细胞和分子水平上研究生物体内生化进程为目标的新兴学科——分子影像学(Molecular imaging,MI)。MI的最显著特点是借助各种分子探针,利用磁共振(Magnetic resonance image, MRI)、光学、核素和超声等影像学技术,实现对单个细胞、乃至单个分子的非侵入性显像。作为生命科学研究的有力工具,分子影像自诞生之初就广泛应用于重大疾病研究、药物研发、干细胞治疗(示踪)等生物医学研究领域,并发挥着越来越重要的作用。而作为MI最为关键的环节,分子成像探针的研发也一直处于分子影像研究的最前沿,并取得了令人瞩目的成就。
然而,随着生命科学研究的不断深入,单纯依靠一种成像方法已经不能满足人们对MI成像精准度的要求。因此,将多种成像模式的分子探针进行融合,开发能同时满足多种成像模式需求的分子探针成为了广大分子影像研究工作者关注的焦点。在MI传统的成像方法中,MRI和光学成像以其无创、实时、无X线电离辐射和可重复性高等优势一直是国内外学者研究的热点,也是最具发展前景的成像手段。MRI和光学探针的融合,不仅可以提高探针的敏感度,而且能够提供靶器官详细的软组织解剖细节,满足早期、动态、实时、无创和精准成像的要求。
在本项目中我们针对当前胶质瘤研究的热点问题,利用具备良好生物相容性的介孔二氧化硅(Mesoporous silica nanoparticles, MSNs)材料、有机近红外荧光染料(IR-808)和磁共振含钆对比剂(Gd-DTPA)构建具有近红外(Near infared, NIR)光学和磁共振双模态成像功能的分子探针,并建立人胶质母细胞瘤皮下肿瘤模型,探讨所构建分子探针的稳定性、靶向成像的效果和生物相容性。以期通过构建的双模态成像分子探针,为胶质瘤的早期诊断提供新的技术支持。
研究目的
一、探讨近红外花菁类染料IR-808对胶质瘤、乳腺癌等恶性肿瘤靶向成像的可行性,并对IR-808的生物相容性进行研究,为后期双模态成像探针的构建寻找合适的NIR光学成像探针；
二、构建具有良好单分散性、高比表面、规则孔道结构和生物相容性的介孔二氧化硅纳米材料,为进一步构建具有MRI和NIR双模态成像的分子探针提供生物纳米材料载体；
三、构建具有良好水溶性、生物相容性的MRI和NIR多功能双模态成像探针,并检验其在体外和活体组织内成像的可行性；
四、探讨所构建的多功能双模态探针对人胶质母细胞瘤(U87-MG)裸鼠模型成像的可行性及其应用价值。
研究方法、内容和结果
一、近红外花菁类染料IRR-808对胶质瘤的靶向性光学成像研究
1、肿瘤细胞培养和标记
C6细胞的培养采用含胎牛血清(15%)、马血清(2.5%)和1%双抗(100U/ml青霉素和100mg/ml链霉素)的F12Kaighn's培养基,人胶质母细胞瘤细胞U87-MG和人乳腺癌细胞MDA-MB-231的培养采用含10%胎牛血清和1%双抗的高糖DMEM培养基；所有细胞均在细胞培养箱内以37摄氏度(℃)、5%CO2及饱和湿度条件下的连续培养传代,所有细胞均贴壁生长。24孔板每孔中加入1μlIR-808溶液(10mM)在370C细胞培养箱内孵育10-15min。多聚甲醛固定后,进行DAPI染色。荧光倒置相差显微镜观察显示：IR-808在体外显示出对大鼠胶质瘤C6细胞、人胶质母细胞U87-MG均具有良好的靶向性：荧光显微镜照片显示,IR-808均匀分布于C6、U87-MG和MDA-MB-231细胞体内,胞核未见近红外荧光染色；而人胚肾细胞HEK293T胞体呈微弱近红外荧光。
2、IR-808的细胞毒性试验
人胚肾细胞HEK293T采用含10%胎牛血清和1%双抗的高糖DMEM培养基；在细胞培养箱内以37℃、5%CO2及饱和湿度条件下的连续培养传代,HEK293T细胞贴壁生长。MTT法计算IR-808的细胞毒性表明正常剂量浓度(20μmol/L)的IR808溶液在共孵育48h时对人胚肾细胞HEK293T的生长无明显影响,2倍于正常浓度的IR808溶液在48h后明显降低了HEK293T细胞的增殖。
3、近红外花菁类染料IR-808靶向MDA-MB-231乳腺癌和C6胶质瘤的活体实验
当裸鼠皮下肿瘤直径达约0.5cm时,经尾静脉注射200μ1IR-808溶液(0.05mmM)。24h后进行光学成像检测,激发波长为778nm,发射光波长为808nm。
乳腺癌肿瘤部位有较明显的近红外荧光信号,且具有良好的背景噪声比。将肿瘤组织完整切除后,体外近红外荧光成像也显示出较高的近红外信号。
BALB/C裸小鼠胶质瘤原位模型活体近红外荧光成像显示肿瘤接种部位有较强的荧光信号,荧光强度可以穿透裸小鼠的颅骨；而SD大鼠活体不能观察到光学信号。处死动物进行脑组织体外成像时,见BALB/C裸小鼠和SD大鼠脑组织肿瘤组织均有较强的近红外荧光。
二、基于带氨基介孔二氧化硅纳米颗粒磁共振和光学双模态探针的制备
1、介孔二氧化硅纳米颗粒的制备和表征
根据参考文献的方法,0.4g十六烷基三甲基溴化铵溶于567.5ml去离子水中,加入3.52ml乙酸乙酯和12.12ml氨水,加入0.9ml正硅酸四乙脂和1.1ml(3-氨丙基)三乙氧基硅烷。将混合液在室温下以300转/分(rpm)充分搅拌反应24小时(h)。去离子水清洗3次,无水乙醇清洗1次。在样品中加入120ml无水乙醇和240μl浓盐酸,在60摄氏度水浴锅中搅拌反应3h。去离子水清洗3次,定容至5ml。
场发射扫描电子显微镜的照片使用Hitachi S4800型扫描电子显微镜拍摄,加速电压为1kV。透射电子显微镜(TEM)的照片使用日本JEOL公司JEM-2100型透射电子显微镜拍摄,加速电压为200kV。通过测量约100个纳米颗粒的直径,计算得到MSNs-NH2的平均粒径为120nm(60～160nm)。氮气吸附一脱附等温线使用北京金埃谱科技有限公司V-Sorb2800P比表面积及孔径分析仪在-196℃测试,测试前将样品在13,200rpm离心10min后,在70℃烘箱内彻底干燥,研磨成均匀粉末状,并在180℃条件下脱气6小时。采用Brunauer-Emmett-Teller (BET)方法在吸附曲线p/p0=0.05～0.15处计算样品的比表面积为～473m2/g；采用Barrett-Joyner-Halenda (BJH)分析法基于吸附数据计算MSNs-NH2的孔径为3nm。介孔二氧化硅材料的傅立叶变换红外光谱使用美国Thermo Nicolet公司NEXUS870型傅立叶红外光谱仪测定。元素分析使用德国Elementar公司Vario MICRO型元素分析仪表明MSNs-NH2的质量百分含量为2.67%。使用英国Malvern公司Nano-Z型Zeta电位仪分析定容后MSNs-NH2的Zeta电位为+32mV。
2、MSNs-Gd/NIR双模态成像分子探针的构建和表征
0.7mg近红外花菁类染料IR-808和4ml0.1摩尔每升的Gd-DTPA溶液加入20ml MES缓冲液(0.1M)中；加入N-羟基琥珀酰亚胺磺酸钠盐0.4mg,室温下避光搅拌2h；然后加入1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐0.6mg,室温下避光搅拌活化反应3-4h。加入1ml介孔二氧化硅溶液,避光反应过夜。
使用美国Thermo Nicolet公司NEXUS870傅立叶红外光谱仪分析介孔二氧化硅修饰IR-808和Gd-DTPA前后的近红外光谱。紫外-可见吸收光谱检测表明IR-808与MSNs-NH2的连接系数为0.3%；美国PE公司OPTIMA5300DV型电感耦合等离子体发射光谱仪ICP显示MSNs-Gd/NIR中钆的浓度为0.009M；不同浓度梯度Gd MR成像结果显示MSNs-Gd/NIR比市售的Gd-DTPA具有更高的弛豫率,能更加显著的缩短T1弛豫时间。使用英国Malvern公司Nano-Z型Zeta电位仪分析定容后MSNs-Gd/NIR的Zeta电位为+7.35mV.
3、MSNs-Gd/NIR的细胞毒性试验
人胚肾细胞HEK293T采用含10%胎牛血清和1%双抗的高糖DMEM培养基；在细胞培养箱内以37℃、5%CO2及饱和湿度条件下的连续培养传代。MTT检测MSNs-Gd/NIR对HEK293T细胞的生长无明显抑制作用。
三、MSNs-Gd/NIR靶向人胶质母细胞瘤U87-MG的体外细胞及荷瘤裸鼠活体试验研究
1、肿瘤细胞对MSNs-Gd/NIR的摄取
吸取100μl前面所制备的MSNs-Gd/NIR溶液(50mg/ml),13,000rpm离心10min后,加入1ml DMEM培养基,超声充分溶解后,分别取20μl加入24孔板内,每孔MSNs-Gd/NIR的终浓度为100μg/ml。在37℃细胞培养箱内孵育2～3h。使用PBS清洗细胞3次,每孔加入300μl多聚甲醛固定30min; PBS再次清洗细胞3次；每孔加入300μl DAPI溶液孵育20min。弃去染色液,PBS清洗1次,在载玻片上滴加1滴甘油,将盖玻片倒扣于载玻片上,指甲油封片。使用Olympus荧光倒置相差显微镜观察细胞的染色情况。结果表明C6胶质瘤细胞和U87-MG-GFP细胞内均有较强的近红外荧光。
2、大鼠神经胶质瘤细胞C6的MRI和NIR体外成像
在C6细胞培养瓶内加入一定量的MSNs-Gd/NIR(终浓度为200μg/ml),对照组C6细胞培养瓶内加入同等量的DMEM培养基；两组细胞均在37℃细胞培养箱内孵育4h后,进行MRI和NIR光学成像。结果显示MSNs-Gd/NIR组C6细胞的T1值明显低于对照组。
3、MSNs-Gd/NIR靶向胶质瘤的活体实验
在人胶质母细胞瘤U87-MG裸鼠皮下肿瘤模型建立成功后,向肿瘤内注射50μ1MSNs-Gd/NIR溶液(5mg/ml),分别在注射后0、1、4、8、12、24和48h后分别行MR和近红外光学检查,在48h后脱颈处死动物,并取肿瘤组织和各重要脏器进行近红外荧光成像。
MRI扫描用德国SIMENS公司MAGNETOM Trio3.0T磁共振扫描仪和定制小动物线圈(4通道)；扫描具体参数如下：横断位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。小动物活体成像仪光学检查使用美国Xenogen公司的IVIS Lumina XR系统,激发波长为778nm,发射光波长为808nm。
在肿瘤内注射50μlMSNs-Gd/NIR溶液(5mg/ml)后,近红外荧光和MR动态观察肿瘤信号的改变。在瘤内注射48小时,肿瘤内仍然能够观察到明显的近红外荧光信号,在MR T1WI上肿瘤内部可以观察到稍高于正常肿瘤组织的信号,范围较前扩大。在完成MR和NIR荧光扫描后,处死动物。取心、脑、肺、肝脏、脾脏、肾脏、小肠和皮肤进行近红外荧光检测,结果显示：肿瘤组织内有较为均匀的近红外荧光显示。
研究结论
1、近红外花菁类染料IR-808具有靶向胶质瘤的作用,且具有良好的生物相容性,有望作为胶质瘤近红外光学成像的分子探针。
2、我们所制备的带氨基的介孔二氧化硅材料具有较大的比表面积、规则的孔道、良好的单分散性和水溶性,有望作为构建多模态成像和诊断治疗学探针的载体。
3、我们所构建的近红外光学和磁共振双模态分子探针MSNs-Gd/NIR具有稳定的光学量子输出和显著的T1弛豫率,生物相容性良好,且非特异性的靶向胶质瘤。有望作为胶质瘤研究的多模态成像工具,用于肿瘤的早期诊断和药物疗效监测。
研究的主要创新点
1、有学者发现新型近红外花菁类染料IR-808能够对肺癌、宫颈癌等多种上皮组织来源的恶性肿瘤进行靶向NIR成像,但目前尚未见有利用IR-808对胶质瘤进行成像研究的报道。本项目率先将IR-808应用于BABL/c裸小鼠原位胶质瘤的光学成像研究,发现IR-808的NIR信号可以穿透BABL/c裸小鼠的颅骨,实现原位胶质瘤的非侵入性NIR成像,显示了良好的研究和应用前景。
2、以往虽然有文献报道利用有机染料和含钆对比剂制备MRI/NIR双模态纳米探针,但其探针的载体制备方法较为繁琐。本项目制备了表面带有大量氨基的介孔二氧化硅纳米颗粒,无需表面修饰即可通过羧合反应连接新型近红外探针IR-808和市售的Gd-DTPA,构建了具有较高T1弛豫率、稳定近红外量子输出、较长成像时间效果的双模态探针；由于作为探针载体的介孔二氧化硅具有规则的孔道结构,因此有望同时装载药物,进一步构建兼具诊断和治疗作用的多功能诊断治疗学探针,实现诊疗一体化。
Background
Gliomas 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.
Purpose
1、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 imaging
1、Cell lines and cell culture
C6cells 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 study
To 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 mice
C6and 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 probe
1、Synthesis and Characterization of MSNs-NH2
Ethyl 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/NIR
0.7mg 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 study
To 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 imaging
1、Cell uptake study using Gd/NIR-MSN
U87-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 mice
U87-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.
Conclusions
1、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 points
1、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.
胶质瘤是中枢神经系统最常见的原发性肿瘤,其发病率高,预后差。胶质瘤中约80%的病变为恶性,主要包括胶质母细胞瘤和间变性胶质瘤。虽然近年来出现了以手术为主,联合放疗、化疗的综合治疗方案,和一些针对肿瘤血管新生的靶向治疗药物,但恶性胶质瘤患者的5年生存率仍然没有得到显著改善。部分胶质瘤患者在接受治疗后还出现肿瘤复发进展、恶性程度增高的现象。这主要是因为恶性胶质瘤特别是胶质母细胞瘤呈浸润性生长,肿瘤细胞沿神经纤维和毛细血管侵犯生长,与正常脑组织、神经纤维和血管分界不清,手术难以彻底切除。此外,手术和放疗区域大量增生的神经胶质,也给残留和复发肿瘤组织的检测带来了很大困难。因此对胶质瘤进行非侵入性的早期诊断、准确定位和边界显示,并区分正常脑组织、肿瘤和增生的神经胶质,对治疗和预后具有十分重要的意义。目前胶质瘤的诊断主要依靠影像学手段,特别是磁共振和核素扫描。但磁共振和核素检查对体积较小、血脑屏障破坏较轻的肿瘤检测的敏感度和特异度较低。如何在提供详细解剖学细节的基础上实现对胶质瘤高敏感度、高特异度的非侵入性的显像,成为全世界范围内研究人员和医务工作者面临的巨大挑战。
随着科技的进步和生命科学的发展,在最近几十年出现了以能够在活体状态下利用影像学方法在细胞和分子水平上研究生物体内生化进程为目标的新兴学科——分子影像学(Molecular imaging,MI)。MI的最显著特点是借助各种分子探针,利用磁共振(Magnetic resonance image, MRI)、光学、核素和超声等影像学技术,实现对单个细胞、乃至单个分子的非侵入性显像。作为生命科学研究的有力工具,分子影像自诞生之初就广泛应用于重大疾病研究、药物研发、干细胞治疗(示踪)等生物医学研究领域,并发挥着越来越重要的作用。而作为MI最为关键的环节,分子成像探针的研发也一直处于分子影像研究的最前沿,并取得了令人瞩目的成就。
然而,随着生命科学研究的不断深入,单纯依靠一种成像方法已经不能满足人们对MI成像精准度的要求。因此,将多种成像模式的分子探针进行融合,开发能同时满足多种成像模式需求的分子探针成为了广大分子影像研究工作者关注的焦点。在MI传统的成像方法中,MRI和光学成像以其无创、实时、无X线电离辐射和可重复性高等优势一直是国内外学者研究的热点,也是最具发展前景的成像手段。MRI和光学探针的融合,不仅可以提高探针的敏感度,而且能够提供靶器官详细的软组织解剖细节,满足早期、动态、实时、无创和精准成像的要求。
在本项目中我们针对当前胶质瘤研究的热点问题,利用具备良好生物相容性的介孔二氧化硅(Mesoporous silica nanoparticles, MSNs)材料、有机近红外荧光染料(IR-808)和磁共振含钆对比剂(Gd-DTPA)构建具有近红外(Near infared, NIR)光学和磁共振双模态成像功能的分子探针,并建立人胶质母细胞瘤皮下肿瘤模型,探讨所构建分子探针的稳定性、靶向成像的效果和生物相容性。以期通过构建的双模态成像分子探针,为胶质瘤的早期诊断提供新的技术支持。
研究目的
一、探讨近红外花菁类染料IR-808对胶质瘤、乳腺癌等恶性肿瘤靶向成像的可行性,并对IR-808的生物相容性进行研究,为后期双模态成像探针的构建寻找合适的NIR光学成像探针；
二、构建具有良好单分散性、高比表面、规则孔道结构和生物相容性的介孔二氧化硅纳米材料,为进一步构建具有MRI和NIR双模态成像的分子探针提供生物纳米材料载体；
三、构建具有良好水溶性、生物相容性的MRI和NIR多功能双模态成像探针,并检验其在体外和活体组织内成像的可行性；
四、探讨所构建的多功能双模态探针对人胶质母细胞瘤(U87-MG)裸鼠模型成像的可行性及其应用价值。
研究方法、内容和结果
一、近红外花菁类染料IRR-808对胶质瘤的靶向性光学成像研究
1、肿瘤细胞培养和标记
C6细胞的培养采用含胎牛血清(15%)、马血清(2.5%)和1%双抗(100U/ml青霉素和100mg/ml链霉素)的F12Kaighn's培养基,人胶质母细胞瘤细胞U87-MG和人乳腺癌细胞MDA-MB-231的培养采用含10%胎牛血清和1%双抗的高糖DMEM培养基；所有细胞均在细胞培养箱内以37摄氏度(℃)、5%CO2及饱和湿度条件下的连续培养传代,所有细胞均贴壁生长。24孔板每孔中加入1μlIR-808溶液(10mM)在370C细胞培养箱内孵育10-15min。多聚甲醛固定后,进行DAPI染色。荧光倒置相差显微镜观察显示：IR-808在体外显示出对大鼠胶质瘤C6细胞、人胶质母细胞U87-MG均具有良好的靶向性：荧光显微镜照片显示,IR-808均匀分布于C6、U87-MG和MDA-MB-231细胞体内,胞核未见近红外荧光染色；而人胚肾细胞HEK293T胞体呈微弱近红外荧光。
2、IR-808的细胞毒性试验
人胚肾细胞HEK293T采用含10%胎牛血清和1%双抗的高糖DMEM培养基；在细胞培养箱内以37℃、5%CO2及饱和湿度条件下的连续培养传代,HEK293T细胞贴壁生长。MTT法计算IR-808的细胞毒性表明正常剂量浓度(20μmol/L)的IR808溶液在共孵育48h时对人胚肾细胞HEK293T的生长无明显影响,2倍于正常浓度的IR808溶液在48h后明显降低了HEK293T细胞的增殖。
3、近红外花菁类染料IR-808靶向MDA-MB-231乳腺癌和C6胶质瘤的活体实验
当裸鼠皮下肿瘤直径达约0.5cm时,经尾静脉注射200μ1IR-808溶液(0.05mmM)。24h后进行光学成像检测,激发波长为778nm,发射光波长为808nm。
乳腺癌肿瘤部位有较明显的近红外荧光信号,且具有良好的背景噪声比。将肿瘤组织完整切除后,体外近红外荧光成像也显示出较高的近红外信号。
BALB/C裸小鼠胶质瘤原位模型活体近红外荧光成像显示肿瘤接种部位有较强的荧光信号,荧光强度可以穿透裸小鼠的颅骨；而SD大鼠活体不能观察到光学信号。处死动物进行脑组织体外成像时,见BALB/C裸小鼠和SD大鼠脑组织肿瘤组织均有较强的近红外荧光。
二、基于带氨基介孔二氧化硅纳米颗粒磁共振和光学双模态探针的制备
1、介孔二氧化硅纳米颗粒的制备和表征
根据参考文献的方法,0.4g十六烷基三甲基溴化铵溶于567.5ml去离子水中,加入3.52ml乙酸乙酯和12.12ml氨水,加入0.9ml正硅酸四乙脂和1.1ml(3-氨丙基)三乙氧基硅烷。将混合液在室温下以300转/分(rpm)充分搅拌反应24小时(h)。去离子水清洗3次,无水乙醇清洗1次。在样品中加入120ml无水乙醇和240μl浓盐酸,在60摄氏度水浴锅中搅拌反应3h。去离子水清洗3次,定容至5ml。
场发射扫描电子显微镜的照片使用Hitachi S4800型扫描电子显微镜拍摄,加速电压为1kV。透射电子显微镜(TEM)的照片使用日本JEOL公司JEM-2100型透射电子显微镜拍摄,加速电压为200kV。通过测量约100个纳米颗粒的直径,计算得到MSNs-NH2的平均粒径为120nm(60～160nm)。氮气吸附一脱附等温线使用北京金埃谱科技有限公司V-Sorb2800P比表面积及孔径分析仪在-196℃测试,测试前将样品在13,200rpm离心10min后,在70℃烘箱内彻底干燥,研磨成均匀粉末状,并在180℃条件下脱气6小时。采用Brunauer-Emmett-Teller (BET)方法在吸附曲线p/p0=0.05～0.15处计算样品的比表面积为～473m2/g；采用Barrett-Joyner-Halenda (BJH)分析法基于吸附数据计算MSNs-NH2的孔径为3nm。介孔二氧化硅材料的傅立叶变换红外光谱使用美国Thermo Nicolet公司NEXUS870型傅立叶红外光谱仪测定。元素分析使用德国Elementar公司Vario MICRO型元素分析仪表明MSNs-NH2的质量百分含量为2.67%。使用英国Malvern公司Nano-Z型Zeta电位仪分析定容后MSNs-NH2的Zeta电位为+32mV。
2、MSNs-Gd/NIR双模态成像分子探针的构建和表征
0.7mg近红外花菁类染料IR-808和4ml0.1摩尔每升的Gd-DTPA溶液加入20ml MES缓冲液(0.1M)中；加入N-羟基琥珀酰亚胺磺酸钠盐0.4mg,室温下避光搅拌2h；然后加入1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐0.6mg,室温下避光搅拌活化反应3-4h。加入1ml介孔二氧化硅溶液,避光反应过夜。
使用美国Thermo Nicolet公司NEXUS870傅立叶红外光谱仪分析介孔二氧化硅修饰IR-808和Gd-DTPA前后的近红外光谱。紫外-可见吸收光谱检测表明IR-808与MSNs-NH2的连接系数为0.3%；美国PE公司OPTIMA5300DV型电感耦合等离子体发射光谱仪ICP显示MSNs-Gd/NIR中钆的浓度为0.009M；不同浓度梯度Gd MR成像结果显示MSNs-Gd/NIR比市售的Gd-DTPA具有更高的弛豫率,能更加显著的缩短T1弛豫时间。使用英国Malvern公司Nano-Z型Zeta电位仪分析定容后MSNs-Gd/NIR的Zeta电位为+7.35mV.
3、MSNs-Gd/NIR的细胞毒性试验
人胚肾细胞HEK293T采用含10%胎牛血清和1%双抗的高糖DMEM培养基；在细胞培养箱内以37℃、5%CO2及饱和湿度条件下的连续培养传代。MTT检测MSNs-Gd/NIR对HEK293T细胞的生长无明显抑制作用。
三、MSNs-Gd/NIR靶向人胶质母细胞瘤U87-MG的体外细胞及荷瘤裸鼠活体试验研究
1、肿瘤细胞对MSNs-Gd/NIR的摄取
吸取100μl前面所制备的MSNs-Gd/NIR溶液(50mg/ml),13,000rpm离心10min后,加入1ml DMEM培养基,超声充分溶解后,分别取20μl加入24孔板内,每孔MSNs-Gd/NIR的终浓度为100μg/ml。在37℃细胞培养箱内孵育2～3h。使用PBS清洗细胞3次,每孔加入300μl多聚甲醛固定30min; PBS再次清洗细胞3次；每孔加入300μl DAPI溶液孵育20min。弃去染色液,PBS清洗1次,在载玻片上滴加1滴甘油,将盖玻片倒扣于载玻片上,指甲油封片。使用Olympus荧光倒置相差显微镜观察细胞的染色情况。结果表明C6胶质瘤细胞和U87-MG-GFP细胞内均有较强的近红外荧光。
2、大鼠神经胶质瘤细胞C6的MRI和NIR体外成像
在C6细胞培养瓶内加入一定量的MSNs-Gd/NIR(终浓度为200μg/ml),对照组C6细胞培养瓶内加入同等量的DMEM培养基；两组细胞均在37℃细胞培养箱内孵育4h后,进行MRI和NIR光学成像。结果显示MSNs-Gd/NIR组C6细胞的T1值明显低于对照组。
3、MSNs-Gd/NIR靶向胶质瘤的活体实验
在人胶质母细胞瘤U87-MG裸鼠皮下肿瘤模型建立成功后,向肿瘤内注射50μ1MSNs-Gd/NIR溶液(5mg/ml),分别在注射后0、1、4、8、12、24和48h后分别行MR和近红外光学检查,在48h后脱颈处死动物,并取肿瘤组织和各重要脏器进行近红外荧光成像。
MRI扫描用德国SIMENS公司MAGNETOM Trio3.0T磁共振扫描仪和定制小动物线圈(4通道)；扫描具体参数如下：横断位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。小动物活体成像仪光学检查使用美国Xenogen公司的IVIS Lumina XR系统,激发波长为778nm,发射光波长为808nm。
在肿瘤内注射50μlMSNs-Gd/NIR溶液(5mg/ml)后,近红外荧光和MR动态观察肿瘤信号的改变。在瘤内注射48小时,肿瘤内仍然能够观察到明显的近红外荧光信号,在MR T1WI上肿瘤内部可以观察到稍高于正常肿瘤组织的信号,范围较前扩大。在完成MR和NIR荧光扫描后,处死动物。取心、脑、肺、肝脏、脾脏、肾脏、小肠和皮肤进行近红外荧光检测,结果显示：肿瘤组织内有较为均匀的近红外荧光显示。
研究结论
1、近红外花菁类染料IR-808具有靶向胶质瘤的作用,且具有良好的生物相容性,有望作为胶质瘤近红外光学成像的分子探针。
2、我们所制备的带氨基的介孔二氧化硅材料具有较大的比表面积、规则的孔道、良好的单分散性和水溶性,有望作为构建多模态成像和诊断治疗学探针的载体。
3、我们所构建的近红外光学和磁共振双模态分子探针MSNs-Gd/NIR具有稳定的光学量子输出和显著的T1弛豫率,生物相容性良好,且非特异性的靶向胶质瘤。有望作为胶质瘤研究的多模态成像工具,用于肿瘤的早期诊断和药物疗效监测。
研究的主要创新点
1、有学者发现新型近红外花菁类染料IR-808能够对肺癌、宫颈癌等多种上皮组织来源的恶性肿瘤进行靶向NIR成像,但目前尚未见有利用IR-808对胶质瘤进行成像研究的报道。本项目率先将IR-808应用于BABL/c裸小鼠原位胶质瘤的光学成像研究,发现IR-808的NIR信号可以穿透BABL/c裸小鼠的颅骨,实现原位胶质瘤的非侵入性NIR成像,显示了良好的研究和应用前景。
2、以往虽然有文献报道利用有机染料和含钆对比剂制备MRI/NIR双模态纳米探针,但其探针的载体制备方法较为繁琐。本项目制备了表面带有大量氨基的介孔二氧化硅纳米颗粒,无需表面修饰即可通过羧合反应连接新型近红外探针IR-808和市售的Gd-DTPA,构建了具有较高T1弛豫率、稳定近红外量子输出、较长成像时间效果的双模态探针；由于作为探针载体的介孔二氧化硅具有规则的孔道结构,因此有望同时装载药物,进一步构建兼具诊断和治疗作用的多功能诊断治疗学探针,实现诊疗一体化。
Background
Gliomas 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.
Purpose
1、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 imaging
1、Cell lines and cell culture
C6cells 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 study
To 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 mice
C6and 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 probe
1、Synthesis and Characterization of MSNs-NH2
Ethyl 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/NIR
0.7mg 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 study
To 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 imaging
1、Cell uptake study using Gd/NIR-MSN
U87-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 mice
U87-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.
Conclusions
1、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 points
1、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.
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71. Akhter S, Ahmad MZ, Ahmad FJ, et al. Gold nanoparticles in theranostic oncology:current state-of-the-art. Expert Opin Drug Deliv 2012;9:1225-43.
72. Heo DN, Yang DH, Moon HJ, et al. Gold nanoparticles surface-functionalized with paclitaxel drug and biotin receptor as theranostic agents for cancer therapy. Biomaterials 2012;33:856-66.
73. Patra CR, Bhattacharya R, Mukhopadhyay D, et al. Fabrication of gold nanoparticles for targeted therapy in pancreatic cancer. Adv Drug Deliv Rev 2010;62:346-61.
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76. Wang X, Wang C, Cheng L, et al. Noble metal coated single-walled carbon nanotubes for applications in surface enhanced Raman scattering imaging and photothermal therapy. J Am Chem Soc 2012;134:7414-22.
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78. Vivero-Escoto JL, Huxford-Phillips RC, Lin W. Silica-based nanoprobes for biomedical imaging and theranostic applications. Chem Soc Rev 2012;41:2673-85.
79. Souris JS, Lee CH, Cheng SH, et al. Surface charge-mediated rapid hepatobiliary excretion of mesoporous silica nanoparticles. Biomaterials 2010;31:5564-74.
80. Kathryn M. L. Taylor JSK, William J. Rieter, Hongyu An, Weili Lin, and Wenbin Lin. Mesoporous Silica Nanospheres as Highly Efficient MRI Contrast Agents. J Am Chem Soc 2008;130:2154-5.
81. Shen Y, Shao Y, He H, et al. Gadolinium(3+)-doped mesoporous silica nanoparticles as a potential magnetic resonance tracer for monitoring the migration of stem cells in vivo. Int J Nanomedicine 2013;8:119-27.
82. Huang X, Zhang F, Lee S, et al. Long-term multimodal imaging of tumor draining sentinel lymph nodes using mesoporous silica-based nanoprobes. Biomaterials 2012;33:4370-8.
83. Lu J, Liong M, Li Z, et al. Biocompatibility, biodistribution, and drug-delivery efficiency of mesoporous silica nanoparticles for cancer therapy in animals. Small 2010;6:1794-805.
84. Estorch M, Carrio I. Future challenges of multimodality imaging. Recent Results Cancer Res 2013;187:403-15.
85. Hoffman JM. Can optical molecular imaging techniques with catheter-based approaches be used for disease detection? Radiology 2004;231:609-10.
86. Sumer B, Gao J. Theranostic nanomedicine for cancer. Nanomedicine (Lond) 2008;3:137-40.
87. Mitsunaga M, Ogawa M, Kosaka N, et al. Cancer cell-selective in vivo near infrared photoimmunotherapy targeting specific membrane molecules. Nat Med 2011;17:1685-91.
88. Luo S, Zhang E, Su Y, et al. A review of NIR dyes in cancer targeting and imaging. Biomaterials 2011;32:7127-38.
89. Shi C, Zhu Y, Xie Z, et al. Visualizing human prostate cancer cells in mouse skeleton using bioconjugated near-infrared fluorescent quantum dots. Urology 2009;74:446-51.
90. Beckman WC, Jr., Powers SK, Brown JT, et al. Differential retention of rhodamine 123 by avian sarcoma virus-induced glioma and normal brain tissue of the rat in vivo. Cancer 1987;59:266-70.
91. Kim RB. Organic anion-transporting polypeptide (OATP) transporter family and drug disposition. Eur J Clin Invest 2003;33 Suppl 2:1-5.
92. Zaidi H, Prasad R. Advances in multimodality molecular imaging. J Med Phys 2009;34:122-8.
93. Huang X, Swierczewska M, Choi KY, et al. Multiplex imaging of an intracellular proteolytic cascade by using a broad-spectrum nanoquencher. Angew Chem Int Ed Engl 2012;51:1625-30.
94. Ahmed N, Fessi H, Elaissari A. Theranostic applications of nanoparticles in cancer. Drug Discov Today 2012;17:928-34.
95. Carniato F, Tei L, Cossi M, et al. A chemical strategy for the relaxivity enhancement of Gd(III) chelates anchored on mesoporous silica nanoparticles. Chemistry 2010;16:10727-34.
96. Suteewong T, Sai H, Cohen R, et al. Highly aminated mesoporous silica nanoparticles with cubic pore structure. J Am Chem Soc 2011; 133:172-5.
97. Kircher MF, Mahmood U, King RS, et al. A multimodal nanoparticle for preoperative magnetic resonance imaging and intraoperative optical brain tumor delineation. Cancer Res 2003;63:8122-5.
98. Jeon YH, Kim YH, Choi K, et al. In vivo imaging of sentinel nodes using fluorescent silica nanoparticles in living mice. Mol Imaging Biol 2010;12:155-62.
99. Sharma P, Bengtsson NE, Walter GA, et al. Gadolinium-doped silica nanoparticles encapsulating indocyanine green for near infrared and magnetic resonance imaging. Small 2012;8:2856-68.
100. H. Yang SS, GA. Walter and P. H. Holloway. Gd(III)-Functionalized Fluorescent Quantum Dots as Multimodal Imaging Probes Adv Mater 2006; 18:2890-4.
101. Caravan P. Strategies for increasing the sensitivity of gadolinium based MRI contrast agents. Chem Soc Rev 2006;35:512-23.
102. J. Kim YP, T. Hyeon. Multifunctional nanostructured materials for multimodal imaging, and simultaneous imaging and therapy. Chem Soc Rev 2009;38:372-90.
103. S. Santra RPB, D. Dutta, J. T. Stanley, G. A. Walter, W. Tan, B. M. Moudgil and R. A. Mericle. Synthesis and Characterization of Fluorescent, Radio-Opaque, and Paramagnetic Silica Nanoparticles for Multimodal Bioimaging Applications Adv Mater 2005;17:2165-9.
104. Ma M, Chen H, Chen Y, et al. Au capped magnetic core/mesoporous silica shell nanoparticles for combined photothermo-/chemo-therapy and multimodal imaging. Biomaterials 2012;33:989-98.
105. Huang X, Zhuang J, Teng X, et al. The promotion of human malignant melanoma growth by mesoporous silica nanoparticles through decreased reactive oxygen species. Biomaterials 2010;31:6142-53.
106. Tanaka S, Louis DN, Curry WT, et al. Diagnostic and therapeutic avenues for glioblastoma:no longer a dead end? Nat Rev Clin Oncol 2013;10:14-26.
107. Sugahara T, Korogi Y, Kochi M, et al. Correlation of MR imaging-determined cerebral blood volume maps with histologic and angiographic determination of vascularity of gliomas. AJR Am J Roentgenol 1998;171:1479-86.
108. Law M, Yang S, Wang H, et al. Glioma grading:sensitivity, specificity, and predictive values of perfusion MR imaging and proton MR spectroscopic imaging compared with conventional MR imaging. AJNR Am J Neuroradiol 2003;24:1989-98.
109. Peet AC, Arvanitis TN, Leach MO, et al. Functional imaging in adult and paediatric brain tumours. Nat Rev Clin Oncol 2012;9:700-11.
110.Manzoor AA, Lindner LH, Landon CD, et al. Overcoming limitations in nanoparticle drug delivery:triggered, intravascular release to improve drug penetration into tumors. Cancer Res 2012;72:5566-75.
111.Hensel JA, Flaig TW, Theodorescu D. Clinical opportunities and challenges in targeting tumour dormancy. Nat Rev Clin Oncol 2013; 10:41-51.
112.Chia-Hung Lee S-HC, Yu-Jing Wang, Yu-Ching Chen, Nai-Tzu Chen, Jeffrey Souris, Chin-Tu Chen, Chung-Yuan Mou, Chung-Shi Yang and Leu-Wei Lo. Near-Infrared Mesoporous Silica Nanoparticles for Optical Imaging: Characterization and In Vivo Biodistribution. Adv Funct Mater 2009; 19:215-22.
113.Taylor KM, Kim JS, Rieter WJ, et al. Mesoporous silica nanospheres as highly efficient MRI contrast agents. J Am Chem Soc 2008;130:2154-5.
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