CN114031546A - 一种羟基自由基的光声成像探针及其制备方法和应用 - Google Patents
一种羟基自由基的光声成像探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及到一种羟基自由基的光声成像探针及其制备方法和应用。一种羟基自由基的光声成像探针的制备方法,其中材料份是按份搭配,包括如下步骤:S1:将73‑75mg的0.09‑0.11mmol的IR‑1048加入到双颈烧瓶中,加入4‑6mL无水甲醇,接着用氮气保护,锡纸包裹双颈烧瓶瓶身,室温搅拌4‑6min;S2:将0.07‑0.09mmol的NaBH4溶于1.8‑2.2mL无水甲醇中后,滴加入双颈烧瓶中,滴加完毕后在室温下搅拌25‑35min;S3:然后减压蒸馏除去溶剂,所得固体溶于8‑12mL二氯甲烷和4‑6mL蒸馏水中,剧烈振荡;S4:分别再用4‑6mL二氯甲烷萃取有机层两次,合并后用无水硫酸钠干燥,减压蒸馏除去溶剂。Hydro‑1048能够在体内和体外均与羟基自由基发生灵敏的反应。更重要的是,该探针可用于检测肝炎病发生过程中产生的羟基自由基。
Description
技术领域
本发明涉及生物体特征检测技术领域,尤其涉及到生物体内羟基自由基的光声成像检测技术,具体为一种羟基自由基的光声成像探针及其制备方法和应用。
背景技术
活性氧(ROS)由于在生理和病理中的重要作用而受到广泛关注。其中羟基自由基被认为是氧化应激损伤的主要原因,它几乎攻击所有类型的生物大分子,并造成不可修复的细胞损伤。羟基自由基与炎症、癌症、急性肝炎的形成有关。因此,原位检测羟基自由基具有重要的作用。
近年来,研究者对羟基自由基检测技术的研究也取得了一些进展。例如当前的的检测方法有电子顺磁共振(EPR)和高效液相色谱法(HPLC)。但其操作繁琐,不能在生物体内实时监测。与这些方法相比,荧光成像作为一种无创方法,在体内具有高灵敏和高时空分辨率。然而,荧光成像的有限组织穿透深度阻止了该方法在临床医学成像在深层组织中获得完整信息的能力。参见Kenry,Duan Y,Liu B.Recent Advances of Optical Imaging inthe Second Near-Infrared Window.Advanced Materials,2018,30,1802394及Zhu S,Tian R,Antaris A L,et al.Near-Infrared-II Molecular Dyes for Cancer Imagingand Surgery.Advanced Materials,2019,31,1900321的介绍,因此,开发一种能够在深层生物组织获取特定分子信息的成像方式,这对于羟基自由基的检测是非常可取的。NIR(Near-Infrared,近红外)-II(1000-1700nm)光声分子成像是一种新兴的无创诊断策略,具有高信噪比和大成像深度的特点。光声信号是由目标分子在短脉冲激光照射下吸收光产生的,由于声波在生物组织中的散射远小于光的散射。这种成像策略克服了常见的光学扩散极限。与NRI-Ⅰ(650-900nm)相比,NRI-Ⅱ(1000-1700nm)显著的减少了光散射和衰减,有效的克服了内源性背景干扰,如血红蛋白,黑色素,脂肪和组织液。目前,已经报道了一些NRI-Ⅱ光声探针用于检测各种疾病,例如Qian C,Jiawen C,Zhijuan Y,et al.NIR-II lightactivated photodynamic therapy with protein-capped gold nanoclusters.NanoResearch,2018,11,5657–5669及Wang S,Lin J,Wang T,et al.Recent Advances inPhotoacoustic Imaging for Deep-Tissue Biomedical Applications.Theranostics,2016,6,2394。类风湿性关节炎的检测,参阅Chen J,Qi J,Chen C,et al.Tocilizumab–Conjugated Polymer Nanoparticles for NIR-II Photoacoustic-Imaging-GuidedTherapy of Rheumatoid Arthritis.Advanced Materials,2020,32,2003399.癌症方面的检测,例如Yan H,Chen J,Li Y,et al.Ultrasmall hybrid protein–copper sulfidenanoparticles for targeted photoacoustic imaging of orthotopic hepatocellularcarcinoma with a high signal-to-noise ratio.Biomaterials ence,2018,7,92-103以.上的介绍等。这些探针大多为有机共轭聚合体复合材料和无机纳米材料复合材料,合成制备较为复杂。
发明内容
Kundu等人报道氢氰胺可被活性氧氧化。例如在这篇文章中的报道:Hydrocyanines:A Class of Fluorescent Sensors That Can Image Reactive OxygenSpecies in Cell Culture,Tissue,and In Vivo.Angewandte Chemie,2009,48,299-303,他们运用氧化还原的原理,利用Cy3、Cy5、Cy7、IR-783和ICG构建了各种荧光探针。IR-1048曾作为母体被用于肿瘤探针的合成。例如文章Xiaoqing M,Jiali Z,Zhihong S,etal.Hypoxia-triggered single molecule probe for high-contrast NIR II/PA tumorimaging and robust photothermal therapy.heranostics 2018;8,6025-6034。在本发明中,我们正是通过Kundu报道的原理来制备一种羟基自由基的光声探针(Hydro-1048),并揭示了其应用。
本发明第一方面提供一种羟基自由基的光声成像探针的制备方法,其中材料份是按份搭配,包括如下步骤:
S1:将73-75mg的0.09-0.11mmol的Et-1048加入到双颈烧瓶中,加入4-6mL无水甲醇,接着用氮气保护,锡纸包裹双颈烧瓶瓶身,室温搅拌4-6min;
S2:将0.07-0.09mmol的NaBH4溶于1.8-2.2mL无水甲醇中后滴加入双颈烧瓶中,滴加完毕后在室温下搅拌25-35min;
S3:然后减压蒸馏除去溶剂,所得固体溶于8-12mL二氯甲烷和4-6mL蒸馏水中,剧烈振荡;
S4:分别再用4-6mL二氯甲烷萃取有机层两次,合并后用无水硫酸钠干燥,减压蒸馏除去溶剂。
进一步的,在于步骤S4后还包括步骤S5:产品经柱层析纯化得到红色固体,收集到的产物。
进一步的,Hydro-1048探针的制备方法,其中材料份是按份搭配,包括如下步骤:
S1:将73.9mg的0.1mmol的Et-1048加入到25mL规格的双颈烧瓶中,加入5mL无水甲醇,接着用氮气保护,锡纸包裹双颈烧瓶瓶身,室温搅拌5min;
S2:将0.08mmol的NaBH4溶于2mL无水甲醇中后滴加入双颈烧瓶中,滴加完毕后在室温下搅拌30min;
S3:然后减压蒸馏除去溶剂,所得固体溶于10mL二氯甲烷和5mL蒸馏水中,剧烈振荡;
S4:分别再用5mL二氯甲烷萃取有机层两次,合并后用无水硫酸钠干燥,减压蒸馏除去溶剂。
本发明中的羟基自由基的光声成像探针制备方法,IR-1048被还原形成Hydro-1048时,C=N共轭双键被打断,吸收峰蓝移;当羟基自由基氧化Hydro-1048形成Et-1048时,C=N被恢复,吸收峰红移到近红外二区。
Hydro-1048能够在体内和体外均与羟基自由基发生灵敏的反应。更重要的是,该探针可用于检测肝炎病发生过程中产生的羟基自由基。
本发明第二方面提供一种羟基自由基的光声成像探针,即Hydro-1048探针,其由前述的一种羟基自由基的光声成像探针的制备方法所制成。
本发明方案中,Hydro-1048探针与羟基自由基反应生成Et-1048分子。该分子在近红外二区有稳定的吸收,并且摩尔吸收系数较大,为1.47×105M-1cm-1。其中,NaBH4可将菁中的C=N键还原为C-N键。由于Et-1048与菁的结构相似,与Et-1048相比,Hydro-1048紫外吸收发生蓝移是因为C=N双键被还原成C-N键,导致分子的共轭链被打断。与此同时,C-N也作为羟基自由基的识别点,可以被羟基自由基氧化并转化回C=N键;也就是说,Hydro-1048可以特异性地与羟基自由基反应生Et-1048,提供“开启”信号。
本发明第三方面提供一种Hydro-1048探针的应用,包括光声成像检测羟基自由基的含量,其特征在于包括以下步骤:
S21:将由前述的Hydro-1048探针用DMSO配置成0.8-1.2mM的储备液;
S22:加入不同量的羟基自由基,其中羟基自由基由芬顿反应(Fe2+/H2O2=1/7~1/5,摩尔比)产生;
S23:加入按体积比为1:0.9~1:1.1的2%的吐温溶液和0.1mM的硫酸溶液作为溶剂体;
S24:添加完药品后,在35~40℃的水浴上恒温孵育25~35min,然后测定光声信号强度。
进一步的,所述的Hydro-1048探针的应用,其特征在于:
S21:将Hydro-1048探针用DMSO配置成1mM的储备液;
S22:加入不同量的羟基自由基,其中羟基自由基由芬顿反应(Fe2+/H2O2=1/6,摩尔比)产生;
S23:加入按体积比为1:1的2%的吐温溶液和0.1mM的硫酸溶液作为溶剂体;
S24:添加完药品后,在37℃的水浴上恒温孵育30min,然后测定光声信号强度。
Hydro-1048通过光声技术可用于体外和体内检测羟基自由基的含量,应用于LPS诱导的小鼠肝部产生羟基自由基的监测,为急性肝炎的发病历程研究提供了一种有效的检测方案。Hydro-1048探针作为近红外二区激活型探针,明显提高了信噪比,减小了背景干扰。因此Hydro-1048探针检测羟基自由基的含量是可行的。
本发明的好处和应用效果将在以下实施例中进一步阐述。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解,其中:
图1为本发明的Hydro-1048结构式;
图2为探针Hydro-1048检测羟基自由基的传感机制及Hydro-1048和Et-1048在DMSO中的吸收光谱图;
图3为Hydro-1048溶液中加入不同浓度羟基自由基的吸收光谱图;
图4为Hydro-1048与·OH响应后的光声光谱图;
图5为不同浓度(0.01-1.7μmol)·OH与Hydro-1048(10μmol/L)反应后在1065nm处的光声信号强度与·OH浓度之间的关系图;
图6为光声强度和·OH的浓度之间相关性的线性拟合图;
图7为Hydro-1048与·OH响应光声信号与时间的关系图;
图8为10μM Hydro-1048对各种干扰物质和羟基自由基的响应图(1:1μM·OH;2:100μM Fe3+;3:100μM Fe2+;4:100μM Cu2+;5:10μM NO2 -;6:100μM NO2 -;7:10μM ONOO-;8:100μM ONOO-;9:10μM H2O2;10:100μM H2O2;11:10μM 1O2;12:100μM 1O2;13:10μM O2 ·-;14:100μMO2 ·-;15:10μM ClO-;16:100μM ClO-);
图9为探针Hydro-1048和·OH的氧化产物Et-1048随时间的稳定性图(含20%FBS的PBS(pH=7.4)溶液,37℃下孵育0-140min);
图10为探针Hydro-1048和·OH的氧化产物Et-1048随温度的稳定性图(含2%Tween-80的PBS(pH=7.4)溶液,在30-45℃条件下孵育);
图11为探针Hydro-1048和·OH的氧化产物Et-1048对介质酸度的稳定性图(含2%Tween-80的Hepes溶液中,pH范围为4-10,37℃条件下孵育);
图12为探针Hydro-1048和·OH的氧化产物Et-1048的稳定性图(激光不同击打次数下的光声强度,含2%Tween-80的PBS(pH=7.4)溶液,37℃条件下孵育);
图13为探针Hydro-1048的细胞毒性研究结果图;a:不同浓度的Hydro-1048孵育24h后细胞的存活率(生理盐水组的细胞存活率自定义为100%);b:小鼠体重随时间依赖变化;c:小鼠尾静脉注射生理盐水(对照组)或Hydro-1048一天后主要器官的代表性切片(H&E染色)。
具体实施方式
为了能够更清楚地理解本发明的上述目的、特征和优点,下面结合附图和具体实施方式对本发明进行进一步的详细描述。需要说明的是,在不冲突的情况下,本申请的实施例及实施例中的特征可以相互组合。
在下面的描述中阐述了很多具体细节以便于充分理解本发明,但是,本发明还可以采用其他不同于在此描述的方式来实施,因此,本发明的保护范围并不受下面公开的具体实施测试例的限制。
下面对本发明实施例作进一步的描述。
为了制备Hydro-1048探针和应用,先行准备好以下仪器和试剂:
电喷雾质谱(Bruker HCT),购买于布鲁克道尔顿公司,用于判定有机化合物的结构;
紫外分光光度计(UV-2600),购买于日本岛津公司,用于吸收光谱的测定;
酶标仪(Multiskan Mk3),购买于Thermo Fisher Scientific公司,用于细胞毒性实验的测试;
pH计(PHSJ-4A),购买于上海精密科学仪器有限公司,用于溶液的配置;
BS 110S电子天平(北京赛多利斯天平有限公司),用于药品的称量,电子称用于老鼠质量的称量;
多光谱光声断层扫描成像***(inVision 256-TF),购买于德国iThera Medical公司,用于体外和活体光声成像的测定。
IR-1048购买于Sigma公司;1×PBS购置于北京索莱宝科技有限公司;无水甲醇购买于成都市科隆化学品有限公司;硼氢化钠购买于国药集团化学试剂有限公司,石油醚和二氯甲烷,二甲基亚砜(DMSO);Tween-80,lipopolysaccharide(LPS)购置于上海阿拉丁生化科技股份有限公司;重水购买于上海众巍化学有限公司;过氧化氢,硫酸亚铁,氢氧化纳,硫酸;氯化钠,氯化铜,六水合氯化铁,硝酸钠,亚硝酸钠,超氧化钾,次氯酸钠均购置于西陇科学股份有限公司。所有的实验试剂均可以购买并且无需进一步纯化即可使用。实验所用细胞株:Hela细胞(人***细胞)购置于中国科学院典型培养物保藏委员会细胞库/中国科学院上海生命科学研究院细胞资源中心;所有裸鼠均购于湖南斯莱克景达实验动物有限公司;动物实验经广西师范大学动物伦理委员会(No.20150325-XC)同意批准。实验其他化学试剂均为国产分析纯,实验用水为18.2MΩ·cm。
Hydro-1048探针的制备按照以下方法进行,其中材料份是按份搭配,包括如下步骤:
S1:将73-75mg的0.09-0.11mmol的Et-1048加入到双颈烧瓶中,加入4-6mL无水甲醇,接着用氮气保护,锡纸包裹双颈烧瓶瓶身,室温搅拌4-6min;
S2:将0.07-0.09mmol的NaBH4溶于1.8-2.2mL无水甲醇中后滴加至双颈烧瓶中,滴加完毕后在室温下搅拌25-35min;
S3:然后减压蒸馏除去溶剂,所得固体溶于8-12mL二氯甲烷和4-6mL蒸馏水中,剧烈振荡;
S4:分别再用4-6mL二氯甲烷萃取有机层两次,合并后用无水硫酸钠干燥,减压蒸馏除去溶剂。
进一步地,在于步骤S4后还包括步骤S5:产品经柱层析纯化得到红色固体,收集到的产物。
进一步地,具体步骤中,S1:将73.9mg的0.1mmol的Et-1048加入到25mL规格的双颈烧瓶中,加入5mL无水甲醇,接着用氮气保护,锡纸包裹双颈烧瓶瓶身,室温搅拌5min;
S2:将0.08mmol的NaBH4溶于2mL无水甲醇中后滴加至双颈烧瓶中,滴加完毕后在室温下搅拌30min;
S3:然后减压蒸馏除去溶剂,所得固体溶于10mL二氯甲烷和5mL蒸馏水中,剧烈振荡;
S4:分别再用5mL二氯甲烷萃取有机层两次,合并后用无水硫酸钠干燥,减压蒸馏除去溶剂。
S5:产品经柱层析纯化得到红色固体,收集到的产物即Hydro-1048探针,约是44mg,产率约58%。
为了构建近红外二区的有机小分子探针,需要增长共轭链来实现,但随着共轭链的增长不利于分子的稳定。Et-1048被选择作为目标分子是因为其在二区有稳定的吸收,并且摩尔吸收系数较大,为1.47×105M-1cm-1,其中,NaBH4可将IR-1404分子中的C=N键还原为C-N键。根据文章Hydrocyanines:A Class of Fluorescent Sensors That Can ImageReactive Oxygen Species in Cell Culture,Tissue,and In Vivo.Angewandte Chemie,2009,48,299-303的报道,由于Et-1048与菁的结构相似,于是我们开发制备了如图1所示的Hydro-1048。如图2所示的探针Hydro-1048检测羟基自由基的传感机制及Hydro-1048和Et-1048在DMSO中的吸收光谱图,与Et-1048相比,Hydro-1048吸收发生蓝移是因为C=N双键被还原成C-N键,导致分子的共轭链被打断;与此同时C-N也作为羟基自由基的识别点,可以被羟基自由基氧化并转化回C=N键;也就是说,Hydro-1048可以特异性地与羟基自由基反应生Et-1048,提供“开启”信号。
本发明的效果通过以下检测和验证可以进一步表现出来。
Hydro-1048探针体外细胞毒性试验和体内毒性评估。
通过在Hela细胞上进行MTT(methyl thiazolyl tetrazolium)试验来测试Hydro-1048的细胞毒性。细胞被重载96孔细胞培养板上并在5%的CO2和37℃的条件下孵育24小时。接着往细胞培养板上的空加入不同浓度的Hydro-1048(0.5,1,2,4,8,12,14,16,18,20μM).细胞在5%的CO2和37℃的条件下孵育24小时。然后,把15μL MTT(5mg/mL)加入到每个孔内,并在5%的CO2和37℃的条件下孵育4小时。除去培养基,往每个孔里加入150μL DMSO。使用Multiskan Mk3型酶标仪在570nm波长处检测每个孔的吸光度。存活率=(实验组的OD570nm/对照组的OD570nm)×100%。在体内毒性评估实验中,分别在雌性BALB/c小鼠(18-20g)尾静脉注射200μL PBS和Hydro-1048(30μmol/L),然后在7天里每天测试小鼠体重。在雌性BALB/c小鼠(18-20g)尾静脉注射200μL PBS和Hydro-1048,一天后将其处死,取其主要器官经4%多聚甲醛固定后,进行常规石蜡包埋,切片厚度5μm,经二甲苯及梯度酒精脱蜡水化后,用苏木素染色5min,伊红复染后封片,光学显微镜下观察并采集图像,通过H&E染色组织切片观察组织的病理损伤情况。(缺少毒性效果评估对比数据)。
羟基自由基含量的检测应用的光声成像,
光谱测定,按以下步骤进行:
S21:将上述的Hydro-1048探针用DMSO配置成0.8-1.2mM的储备液用于测定吸收光谱和光声光谱(在吸收光谱实验和光声光谱实验中,探针的最终浓度为10μM);
S22:在测定时,加入不同量的羟基自由基,羟基自由基由芬顿效应(Fe2+/H2O2=1/7~1/5,摩尔比)产生,具体按(Fe2+/H2O2=1/6,摩尔比)产生;
S23:加入按体积比为1:0.9~1:1.1的2%的吐温溶液和0.1mM的硫酸溶液作为溶剂体,具体的,溶剂体系为2%的吐温溶液和0.1mM的硫酸溶液,体积比为1:1;
S24:在添加完药品后,在35~40℃的水浴上恒温孵育25~35min,具体可以选择在37℃的水浴上恒温孵育30min,然后测定光谱。具体的,吸收光谱测定进行基线校正,光声光谱进行背景扣除)。
其他关于ROS的配置均见参考文献――Daihi Oushiki,Hirotatsu Kojima,Takuya Terai,Makoto Arita,Kenjiro Hanaoka,Yasuteru Urano and TetsuoNagano.Development and application of a near-infrared fluorescence probe foroxidative stress based on differential reactivity of linked cyaninedyes..Journal of the American Chemical Society,2010,132,2795-801。
动物实验:羟基自由基在活鼠内的检测。用LPS建立动物模型,8周龄的雌性BALB/c小鼠(~20g)购买于湖南斯莱克景达实验动物有限公司(Hunan SJA Laboratory AnimalCO.Ltd)。建模老鼠腹腔注射0或15mg/Kg的LPS(LPS用生理盐水配置),对照组注射生理盐水。在建模24h后,所有的老鼠尾静脉注射Hydro-1048(5mL/Kg,用2%吐温生理盐水配置,使得最终浓度为1mM)。在注射两小时后,对小鼠麻醉,进行光声成像。各组均用6只小鼠进行重复实验。
Hydro-1048探针与羟基自由基响应的光学特性。
往Hydro-1048溶液中加入不同浓度羟基自由基的吸收光谱(UV-2600)。如图3所示,随着羟基自由基浓度在0.01-1.7μmol/L的范围内增加,Hydro-1048溶液(10μmol/L)在1065nm处的吸收强度(Abs1065)逐渐增强,与此同时在520nm处的吸收强度(Abs520)逐渐下降。溶液颜色由红色变成无色。Hydro-1048与羟基自由基响应后的光声信号变化如图4所示,加入羟基自由基后,Hydro-1048溶液在1065nm处的光声信号极具增加。记录不同浓度羟基自由基与Hydro-1048(10μmol/L)反应后在1065nm处的光声信号如图5所示,发现光声信号和羟基自由基的浓度呈正相关,当羟基自由基的浓度达到1μmol/L以后,光声信号的强度趋于平稳。将其线性拟合,发现在10-220nM(R2=0.9917)和220-1020nM(R2=0.9892)的范围内,光声强度和羟基自由基的浓度呈线性关系,如图6。运用公式3Sb/k(Sb为11个空白样品的标准偏差,k为斜率)计算其最低检测限为0.6nM,可以检测出老鼠肝脏中羟基自由基的含量。具体可以参阅Itoh H,Ohkuwa T,Yamamoto T,et al.Effects of endurance physicaltraining on hydroxyl radical generation in rat tissues.Life Sciences,1998,63,1921-1929。
Hydro-1048探针与羟基自由基响应的时间依赖性,由于羟基自由基的寿命极短,因此探针的响应速率至关重要。为此,考察Hydro-1048探针的时间依赖性,在测定时加入等量的羟基自由基,羟基自由基由芬顿效应(Fe2+/H2O2=1/6,摩尔比)产生。溶剂体系为2%的吐温溶液和0.1mM的硫酸溶液,体积比为1:1。在添加完药品后,在37℃的水浴上恒温孵育不同的时间。得出光声强度和孵育时间的关系,如图7所示。当孵育时间为30min时为羟基自由基的最佳测定时间。
探针Hydro-1048对羟基自由基的选择性,为了考察探针Hydro-1048对羟基自由基的特异性响应,对体内主要干扰性物质进行了实验。干扰物质分别为100μM Fe3+、100μM Fe2 +、100μM Cu2+、10μM NO2 -、100μM NO2 -、10μM ONOO-、100μM ONOO-、10μM H2O2、100μM H2O2、10μM 1O2、100μM 1O2、10μM O2 ·-、100μM O2 ·-、10μM ClO-、100μM ClO-。从图8可以看出,与其他物质相比,只有1,即1μM羟基自由基(·OH)的光声信号最强,可以得出Hydro-1048对羟基自由基的检测具有特异性。该实验的溶剂体系为pH为7.4的PBS溶液含有2%的Tween-80,Hydro-1048浓度为10μM,实验条件为37℃水浴孵育30min,收集的光声信号为1065nm处,其中所有数据已经扣除探针本身背景。
Hydro-1048探针以及氧化产物Et-1048的稳定性。将探针Hydro-1048和Et-1048在含20%FBS的PBS溶液中孵育0-140min(37℃),从图9中可以看出,Hydro-1048和Et-1048在孵育前后的光声信号差异不大,说明探针比较稳定,可用于生物体内。为了探究Hydro-1048和Et-1048的热稳定性,做了热稳定性实验,以PBS(pH=7.4)含2%的Tween-80作为溶剂体系,在30-45℃不同温度下孵育,得出光声信号的温度的关系,如图10所示,可以得出,在该温度下,探针比较稳定。pH在4-10,探针表现得比较稳定,如图11所示。我们还探讨了探针的光稳定性,我们用1065nm激光连续循环照射探针溶液,每个循环照射1min,关闭1min。然后在不同的循环次数下检测探针溶液的PA信号,得到循环次数与PA强度的关系如图12所示的光声强度和照射次数的关系图,从图可以看出,即使是激光连续循环照射2000次之久,探针依然很稳定。
Hydro-1048探针的细胞毒性和体内毒性。进行动物实验之前,充分评估探针Hydro-1048的生物毒性。使用MTT方法考察了Hydro-1048对人正常肝细胞(HL-7702)的细胞毒性。如图13所示,其中a:不同浓度的Hydro-1048孵育24h后细胞的存活率(生理盐水组的细胞存活率自定义为100%);b:小鼠体重随时间依赖变化;c:小鼠尾静脉注射生理盐水(对照组)或Hydro-1048一天后主要器官的代表性切片(H&E染色)。从图中可以知道,Hydro-1048溶液(0.5-20μmol/L)孵育细胞24小时后,细胞存活率没有明显下降(大于87%)。对于探针的体内毒性的评估,通过连续记录一周小鼠的体重和主要器官的H&E染色组织切片,和对照组相比,实验组小鼠体重没有明显的差异。从小鼠主要器官的组织切片来看,对照组和实验组没有明显差异。从以上可以得出,该探针低毒性。
总而言之,通过前述的检测和测试,经过破坏和恢复共轭体系,前述方法制备的Hydro-1048探针,是一个近红外二区羟基自由基的光声探针,可以用于光声成像实时检测体内羟基自由基的含量。Hydro-1048通过光声技术可用于体外和体内检测羟基自由基的含量。
以上显示和描述了本发明的基本原理和主要特征及本发明的优点,本行业的技术人员应该了解,以上仅为本发明的优选测试例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的创造性精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (6)
1.一种羟基自由基的光声成像探针的制备方法,其中材料份是按份搭配,包括如下步骤:
S1:将73-75mg的0.09-0.11mmol的IR-1048加入到双颈烧瓶中,加入4-6mL无水甲醇,接着用氮气保护,锡纸包裹双颈烧瓶瓶身,室温搅拌4-6min;
S2:将0.07-0.09mmol的NaBH4溶于1.8-2.2mL无水甲醇中后滴加入双颈烧瓶中,滴加完毕后在室温下搅拌25-35min;
S3:然后减压蒸馏除去溶剂,所得固体溶于8-12mL二氯甲烷和4-6mL蒸馏水中,剧烈振荡;
S4:分别再用4-6mL二氯甲烷萃取有机层两次,合并后用无水硫酸钠干燥,减压蒸馏除去溶剂。
2.根据权利要求1所述的一种羟基自由基的光声成像探针制备方法,其特征在于步骤S4后还包括步骤S5:产品经柱层析纯化得到红色固体,收集到的产物。
3.根据权利要求1或2所述的一种羟基自由基的光声成像探针制备方法,其特征在于
S1:将73.9mg的0.1mmol的IR-1048加入到25mL规格的双颈烧瓶中,加入5mL无水甲醇,接着用氮气保护,锡纸包裹双颈烧瓶瓶身,室温搅拌5min;
S2:将0.08mmol的NaBH4溶于2mL无水甲醇中后滴加入双颈烧瓶中,滴加完毕后在室温下搅拌30min;
S3:然后减压蒸馏除去溶剂,所得固体溶于10mL二氯甲烷和5mL蒸馏水中,剧烈振荡;
S4:分别再用5mL二氯甲烷萃取有机层两次,合并后用无水硫酸钠干燥,减压蒸馏除去溶剂。
4.一种羟基自由基的光声成像探针,其由权利要求1-3任一项所述的一种羟基自由基的光声成像探针的制备方法所制成。
5.一种羟基自由基的光声成像探针的应用,包括检测羟基自由基的含量,其特征在于包括以下步骤:
S21:将权利要求4所述的一种羟基自由基的光声成像探针用DMSO配置成0.08-1.2mM的储备液;
S22:加入不同量的羟基自由基,其中羟基自由基由芬顿反应(Fe2+/H2O2=1/7~1/5,摩尔比)产生;
S23:加入按体积比为1∶0.9~1∶1.1的2%的吐温溶液和0.1mM的硫酸溶液作为溶剂体;
S24:添加完药品后,在35~40℃的水浴上恒温孵育25~35min,然后测定光声光谱。
6.根据权利要求5所述的一种羟基自由基的光声成像探针的应用,其特征在于:
S21:将Hydro-1048探针用DMSO配置成1mM的储备液;
S22:加入不同量的羟基自由基,其中羟基自由基由芬顿反应(Fe2+/H2O2=1/6,摩尔比)产生;
S23:加入按体积比为1∶1的2%的吐温溶液和0.1mM的硫酸溶液作为溶剂体;
S24:添加完药品后,在37℃的水浴上恒温孵育30min,然后测定光声光谱。
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