CN109293638A - 一种增强型的靶向识别受体酪氨酸激酶的荧光传感器及细胞膜荧光成像的应用 - Google Patents
一种增强型的靶向识别受体酪氨酸激酶的荧光传感器及细胞膜荧光成像的应用 Download PDFInfo
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Abstract
一种增强型的靶向识别受体酪氨酸激酶的荧光传感器及细胞膜荧光成像的应用，其属于生物荧光传感的技术领域。荧光传感器SP1通过舒尼替尼的有效部分作为识别基团，芘作为荧光基团，通过连接基团连接而成。受体酪氨酸激酶作为一种存在于细胞膜上的蛋白，在肿瘤细胞和血管生成过程中大量富集。该荧光传感器SP1可以有效的作用于受体酪氨酸激酶的细胞膜内结构域，相比于细胞中存在的氨基酸及无机盐等干扰物，荧光传感器SP1展示出高效的选择性，对受体酪氨酸激酶具有靶向性的识别效果。SP1对受体酪氨酸激酶的识别具有良好的选择性和较高的灵敏度，能够在细胞、组织及活体内实现对受体酪氨酸激酶的荧光成像，在早期癌症诊断和可视化治疗等领域显示出潜在的应用前景。
Description
技术领域
本发明涉及一种增强型靶向识别受体酪氨酸激酶的荧光传感器及细胞膜荧光成像的应用，其属于生物荧光传感的技术领域。
背景技术
肿瘤靶向治疗癌症的可视化治疗和诊断方面发展迅速。舒尼替尼是一种多靶点的酪氨酸激酶抑制剂，具有抗肿瘤和血管增生的作用，对于许多肿瘤相关的疾病如乳腺癌、肺癌、前列腺癌和结肠直肠癌具有良好的效果。受体酪氨酸激酶位于细胞膜在常见的肿瘤细胞中过表达。通过靶向识别癌细胞体酪氨酸激酶，可以实现癌症的早期诊断及治疗，降低癌症死亡率。
分子荧光成像技术具有生物兼容性好，选择性及分辨率高，损害小，成本低等优点，因此成为一种有前景的肿瘤成像的技术。近期有多种关于与肿瘤相关的荧光传感器的研究，但是用于靶向识别受体酪氨酸激酶的荧光传感器少见报道。
发明内容
为解决现有技术中存在的问题，本发明设计合成了一种增强型的受体酪氨酸激酶的细胞膜荧光传感器SP1，该荧光传感器能够用于在体内和体外对受体酪氨酸激酶进行荧光检测及成像。
本发明采用的技术方案为：一种增强型的靶向识别受体酪氨酸激酶的荧光传感器，该荧光传感器SP1的结构式为：
。
一种增强型的靶向识别受体酪氨酸激酶的荧光传感器的应用，荧光传感器SP1应用于对受体酪氨酸激酶的荧光检测，在0-0.6 μg/mL内，荧光传感器SP1的荧光强度与受体酪氨酸激酶的浓度呈线性关系。
荧光传感器SP1的工作原理为：在游离状态下，SP1以二聚体的形式存在，荧光被淬灭，受体酪氨酸激酶作用后，舒尼替尼有效部分与受体结合， SP1的荧光恢复（如图1）。荧光传感器SP1通过舒尼替尼有效部分作为识别基团，芘作为荧光基团，通过连接基团连接而成。通过NOESY谱图可以发现芘二聚的弱相互作用信号，表明在溶液中，荧光传感器SP1在溶液中以二聚体的形式存在（如图2a）。在DMSO/H2O溶液条件下，SP1因二聚体的存在而呈现较弱的荧光，在加入受体酪氨酸激酶后，SP1的荧光发射增强，表明SP1和受体酪氨酸激酶之间存在一定的相互作用（如图2b）。
本发明的有益效果为：荧光传感器SP1通过舒尼替尼有效部分作为识别基团，芘作为荧光基团，通过连接基团连接而成。受体酪氨酸激酶作为一种存在于细胞膜上的蛋白，在肿瘤细胞和血管生成过程中大量富集。该荧光传感器SP1可以有效的作用于受体酪氨酸激酶的细胞膜内结构域，相比于细胞中存在的氨基酸及无机盐等干扰物，荧光传感器SP1展示出高效的选择性，对受体酪氨酸激酶具有靶向性的识别效果。SP1对受体酪氨酸激酶的识别具有良好选择性和较高的灵敏度，能够在细胞、组织及活体内实现对受体酪氨酸激酶的荧光成像，在早期癌症诊断和可视化治疗等领域显示出潜在的应用前景。
附图说明
图 1. 荧光传感器SP1 识别受体酪氨酸激酶示意图。
图 2. (a) SP1在DMSO-d 6 溶液中的NOESY谱图 (b) 向SP1 （2μM）中加入受体酪氨酸激酶的荧光变化 (c) SP1 在不同干扰物质存在下的荧光响应 (d) CCK8方法测得SP1在HT-29细胞中毒性。
图 3.D4292（1 μM） 和 SP1（0.5 μM）在HT-29 细胞 (a-c), A549细胞(d-f),HUVECs细胞（g-i）中的荧光成像（a，d，g为D4292通道，b，e，h为SP1通道）。
图 4. SP1在HT-29肿瘤细胞中的荧光成像a，0.2 μM；b，0.5 μM；c，1 μM。
图 5. SP1 在HT-29细胞移植瘤裸鼠模型中的荧光成像（a，0.1 mM；b，0.5 mM；c，1mM；d，2 mM）。
具体实施方式
实施例1 荧光传感器SP1的合成
化合物1的合成：
称取5-氟-2,3-吲哚酮 (4.95 g, 30.0 mmol) 和 3 mL联氨（100%）于50 mL n-BuOH，室温下搅拌30 min。80 ℃下加热3 h，加入5 mL 三乙胺，加热至100℃搅拌12 h。冷却至室温，减压蒸馏, 将粗产物溶解至100 mL乙酸乙酯，使用10 % 硫酸氢钾溶液洗涤，萃取。残留物溶解于50 mL热乙酸乙酯中，加入石油醚。在过滤和冷却到室温后，过滤得到棕褐色化合物1（2.84 g, 18.80mmol）。1H NMR (500 MHz, DMSO-d 6 ) δ 10.40 (s, 1H), 7.10 (d, J =8.2 Hz, 1H), 6.99 (t, J = 9.0 Hz, 1H), 6.79 (dd, J = 8.4, 4.5 Hz, 1H), 3.50(s, 2H)。
化合物2的合成：
在10 mLCH2Cl2中加入DMF（0.80 g，11.0 mmol）和磷酰氯(V)（1.69 g，11.0 mmol），冷却到4 ℃后滴加2,4-二甲基-吡咯-3-甲酸乙酯。反应混合物搅拌15分钟并加热1 h。加入10℃的盐酸溶液（5 mL, 10 M），并剧烈搅拌，静置分层。用20 mL盐酸和20 mL CH2Cl2萃取，加入NaOH（25 mL, 10 M）得到黄色固体（1.49 g, 8.0 mmol）。加入90% KOH （20 mL），回流5h。 冷却至室温后，向溶液中加入30 mL水并以40 mL CH2Cl2洗涤。用稀盐酸将溶液pH值调节为4，得到黄色产物（1.14 g, 6.82mmol）。1H NMR (400 MHz, DMSO-d 6 ) δ 12.12 (s, 2H),9.61 (s, 1H), 2.46 (s, 3H), 2.42 (s, 3H).
化合物3的合成：
取化合物1（8.8 mmol, 1.33 g）、2 （8.8 mmol, 1.47 g），四氢化吡咯（18.0 mmol,1.5 mL）在120 mL乙醇中加热回流3 h。冷却至室温，加入15 mL稀盐酸， 过滤，用分别用20mL乙醇和石油醚洗涤得到黄色产物（2.34 g, 7.80 mmol）。1H NMR (400 MHz, DMSO-d 6 ) δ13.82 (s, 1H), 12.11 (s, 1H), 10.87 (s, 1H), 7.74-7.63 (m, 1H), 6.95-6.77 (m,1H), 2.50 (d, J = 10.4 Hz, 1H).
化合物4的合成：
称取化合物3（1.008 g, 3.36 mmol），EDC（1.080 g, 7 mmol），HOBT（616 mg, 4.56mmol），加入2 mL三乙胺和50 mL干燥DMF，30 ℃下搅拌48 h。将混合物倒入5 %的Na2CO3溶液中，静置后进行抽滤。将得到的粗产物使用二氯甲烷：甲醇=500:6进行柱层析分离，得到黄色产物（818 mg, 1.91 mmol)。1H NMR (400 MHz, DMSO-d 6 ) δ 13.65 (s, 1H), 10.86 (s,1H), 7.72 (d, J = 9.3 Hz, 1H), 7.68 (s, 1H), 7.61 (t, J = 5.4 Hz, 1H), 6.89(t, J = 9.0 Hz, 1H), 6.81 (dd, J = 8.3, 4.5 Hz, 1H), 3.56 (s, 3H), 3.18 (dd,J = 12.5, 6.3 Hz, 2H), 2.38 (d, J = 7.0 Hz, 6H), 2.29 (t, J = 7.3 Hz, 2H),1.60- 1.41 (m, 4H), 1.37-1.24 (m, 2H).
化合物5的合成：
称量400 mg化合物4 置于300 mL甲醇并加热至70 ℃，加入60 mL 8 g/L KOH。待溶液温度至室温后，搅拌24 h。向溶液中加入稀盐酸，调节pH=3。抽滤，干燥得到黄色产物（232mg, 0.56mmol）。1H NMR (500 MHz, DMSO-d 6 ) δ 13.67 (s, 1H), 10.88 (s, 1H), 7.76(dd, J = 9.3, 2.2 Hz, 1H), 7.71 (s, 1H), 7.63 (t, J = 5.6 Hz, 1H), 6.92 (td,J = 9.3, 2.4 Hz, 1H), 6.84 (dd, J = 8.4, 4.5 Hz, 1H), 3.59 (s, 1H), 3.21 (dd,J = 12.7, 6.5 Hz, 2H), 2.41 (d, J = 9.4 Hz, 6H), 2.22 (t, J = 7.3 Hz, 2H),1.59-1.47 (m, 4H), 1.33 (dt, J = 14.7, 7.4 Hz, 2H)。
SP1的合成：
称取化合物5（104.5 mg, 0.25mmol），EDC（101 mg, 0.53mmol），HOBT（58 mg,0.43mmol），芘甲基胺盐酸盐（96.37 mg, 0.36mmol）, 加入0.2 mL三乙胺和10 mL干燥DMF，30 ℃下搅拌48 h。将混合物倒入5 %的Na2CO3溶液中，静置后进行抽滤。将得到的粗产物使用二氯甲烷：甲醇=500:6进行柱层析分离，得到黄色产物（35.8mg, 0.057mmol）。1H NMR(400 MHz, DMSO-d 6 ) δ 13.65 (s, 1H), 10.88 (s, 1H), 8.51 (s, 1H), 8.36 (d, J =9.1 Hz, 1H), 8.28 (dt, J = 17.8, 7.6 Hz, 4H), 8.15 (s, 2H), 8.10-8.00 (m,2H), 7.76 (d, J = 8.8 Hz, 1H), 7.70 (s, 1H), 7.62 (s, 1H), 6.93 (t, J = 8.8Hz, 1H), 6.86 (d, J = 4.4 Hz, 1H), 5.01 (d, J = 4.9 Hz, 2H), 3.21 (d, J = 5.9Hz, 2H), 2.41 (d, J = 6.1 Hz, 6H), 2.20 (d, J = 6.7 Hz, 2H), 1.66-1.58 (m,2H), 1.56-1.47 (m, 2H), 1.34 (s, 2H)。
实施例2 荧光传感器SP1的应用
实施方法：
实验试剂：合成SP1所需的各个化合物及测试所需的各种分析物均通过商业渠道购买，无需经过进一步纯化。荧光测试：配制2×10-3 mol/L的探针SP1浓溶液，稀释至荧光比色皿，测试溶液为DMSO/H2O=1:9 (40 mM Tris-HCl, pH=7.4)，荧光激发波长为460 nm，加入受体酪氨酸激酶，使用Edinburgh FS920稳态荧光分光光度计进行荧光测试。
细胞测试：细胞在5 % CO2的37 ℃条件下进行培养，加入SP1后，孵化30 min，经PBS溶液洗涤三次，使用OLYMPUS FV1000激光共聚焦荧光显微镜成像。
小鼠实验：构建结肠癌HT-29裸鼠皮下移植瘤模型，尾静脉注射100 μL不同浓度的SP1，通过小动物活体荧光成像系统在460 nm 激发光下，观察肿瘤部位荧光强度。
通过NOESY谱图可以发现芘环二聚的弱相互作用信号，表明在溶液中，荧光传感器SP1在溶液中以二聚体的形式存在（如图2a）。在DMSO/H2O溶液条件下，SP1因二聚体的存在而呈现较弱的荧光，在加入受体酪氨酸激酶后，SP1的相对荧光发射（F/F 0 ）增强，表明SP1和受体酪氨酸激酶之间存在一定的相互作用（如图2b）。同时在加入细胞中广泛存在的各类氨基酸，无机盐及其他相关的可能干扰物，对SP1识别受体酪氨酸激酶的效果影响微弱（如图2c）。SP1对受体酪氨酸激酶的半数抑制浓度IC50为2.2 ± 0.1 μM，证明了传感器与受体酪氨酸激酶之间具有一定的结合程度。传感器能够在体外选择性地识别受体酪氨酸激酶，并可能在体内对其进行荧光响应。
如图2d，在生物成像试验之前，通过CCK8实验研究了SP1（0-20 μM）对癌细胞的潜在毒性，实验表明荧光传感器对细胞的生物毒性在可接受范围内。HT-29细胞，A549细胞和HUVECs细胞广泛存在于人类的上皮组织中，受体酪氨酸激酶在这三种细胞类型中过度表达。在HT-29细胞，A549细胞和HUVECs细胞中加入1 μM 的一种商业可获得的膜染料D4292和0.5 μM 的SP1孵化30分钟后，可以发现上述三种细胞的细胞膜表面荧光强度显著增高（如图3）。在HT-29细胞中加入SP1（0.2 μM，0.5μM，1 μM），细胞膜上的荧光强度随着探针的浓度增加而增强（如图4）。SP1标记与D4292标记细胞的荧光成像信号基本重合，荧光传感器SP1可以靶向识别受体酪氨酸激酶，在相应细胞的细胞膜上成像。
构建结肠癌HT-29裸鼠皮下移植瘤模型，如图5所示，将不同浓度的SP1直接对裸鼠进行注射，注射后小鼠肿瘤部位出现强烈的荧光信号，并随着SP1浓度增加而增强，只有肿瘤部位显示出强烈的荧光图像，其他部位没有明显荧光信号。结果表明，传感器SP1能够实现在活体肿瘤中对受体酪氨酸激酶的荧光成像。
Claims (2)
1.一种增强型的靶向识别受体酪氨酸激酶的荧光传感器，其特征在于：该荧光传感器SP1的结构式为：
。
2.根据权利要求1所述的一种增强型的靶向识别受体酪氨酸激酶的荧光传感器的应用，其特征在于：荧光传感器SP1应用于对受体酪氨酸激酶的荧光检测，荧光传感器SP1在0-0.6 μg/mL内定量识别受体酪氨酸激酶，定位响应肿瘤细胞的细胞膜。
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| WO2005053686A1 (en) * | 2003-11-26 | 2005-06-16 | The Scripps Research Institute | Indolinone based protein kinase inhibitors |
| WO2013157021A1 (en) * | 2012-04-20 | 2013-10-24 | Advinus Therapeutics Limited | Bicyclic compounds, compositions and medicinal applications thereof |
| CN104662018A (zh) * | 2012-04-20 | 2015-05-27 | 阿迪维纳斯治疗有限公司 | 取代的杂双环化合物、组合物及其医疗应用 |
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