CN115181052B - 抗耐药菌近红外光治疗分子的制备及应用 - Google Patents
抗耐药菌近红外光治疗分子的制备及应用 Download PDFInfo
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Abstract
本发明公开了一类具有细菌靶向的近红外光敏剂的制备及其用于耐多药幽门螺旋杆菌生物膜光疗。该类光敏剂由预修饰靶向基团的IR780分子与聚集诱导发光试剂TPE结构构成。该类抗菌光敏剂表现出良好的光热、光动及光热稳定性,且生物安全性好。细菌实验表明,该类光敏剂结构上的富正电荷基团具有良好的细菌靶向性。抗菌实验表明,在低浓度(10μM)和低激光功率(808nm,0.3W/cm2)下,该光敏剂所不仅能使浮游幽门螺杆菌细胞膜裂解,还可以破坏已形成的生物膜的完整性并杀伤其中的细菌(附图)。该光敏剂具有良好的光热特性及细菌性,因此有望应用于临床耐药菌的光疗研究中。
Description
技术领域
本发明属于细菌光疗技术领域,具体涉及一类具有细菌靶向和高光热转换效率的光敏剂的制备及其在耐多药幽门螺旋杆菌生物膜光疗中的应用。
背景技术
公开此背景技术的信息仅仅只为了增加对本发明的总体理解,并不暗示或承认此部分背景内容已经成为本领域一般技术人员所公知的技术。
幽门螺杆菌(H.pylori)感染一直是人类健康的一类重大威胁,在世界范围内导致极高的胃部感染率,尤其是在低收入国家。在青霉素于1928年发现后,各种抗生素得到了广泛的发展,包含两种抗生素和一种质子泵抑制剂的三联疗法逐渐成为根除幽门螺杆菌的有效途径。然而,在过去的几十年里,抗生素的滥用引发了幽门螺杆菌耐药性的急剧增加,使得传统的抗生素疗法在治疗常见的细菌感染时不再有效。多重耐药性(MDR)幽门螺杆菌的出现证实了这种威胁,MDR幽门螺杆菌对所有已知的抗生素都具有耐药性。此外,幽门螺杆菌群落还可以嵌入自分泌的细胞外聚合物(EPS)中形成生物膜,包裹在生物膜中的细菌不仅对抗生素的抵抗力提高10-1000倍,而且可以使细菌隔离宿主的免疫反应。因此,迫切需要为耐多药幽门螺杆菌及其生物膜开发新的替代疗法。
近红外(Near-infrared,NIR)响应光疗剂(phototherapy agents,PTAs)作为一种光控抗菌材料很有吸引力,因为它可以有效地将NIR光转化为热能或活性氧物质(reactiveoxygen species,ROS)以消融细菌,并且具有很高的空间和时间精度。更重要的是,光疗(phototherapy,PT)可以通过多种机制克服细菌耐药性。首先,PT是非选择性的,因此细菌很难对它产生抗性。其次,与抗生素不同,药物内化于细胞在PT中不是必需的,从而进一步克服了细菌的耐药性。第三,PTAs还可以与生物膜的主要成分相互作用;因此,它们可以消除由此引起的耐药性。所以,PT是消除威胁生命的病原体的一种很有前途的方法。在PTAs中,有机PTAs(OPTAs)可以在治疗后很快排出体外,无需过多担心长期组织毒性,因此具有广阔的生物学前景。然而,目前大多数OPTAs在水中的溶解性较差,在生理条件下容易聚集,在酸性条件下容易发生光漂白、热降解和氧化,通常需要与其他功能聚合物进行后缀合。因此,开发具有适当水溶性、光稳定性和酸稳定性的新型有机OPTA以满足日益增长的抗菌需求非常重要。
IR780作为一种近红外七甲川花菁染料,广泛应用于肿瘤细胞成像。在我们小组过去的工作中,通过将聚集诱导发光试剂(aggregated-induced emission agents,AIEgens)合理结合到IR780结构中获得的分子显示出改善的光稳定性和光热响应。受此结果的启发,我们尝试将两个IR780与一个AIEgens集成,发现开发的新型“工”字形分子不仅具有更高的光热转换效率和ROS生成能力,而且具有抗光漂白性,热稳定性,在光疗中具有巨大的应用潜力。此外,越来越多的证据表明,带正电荷的多价平台可用作生物膜的破坏剂,因为生物膜的许多成分在生理环境中都带负电荷。其中,富含正电荷的氨基、胍衍生物及氨基苯硼酸由于其抗菌潜力以及膜结合和渗透能力而备受关注。
发明内容
如上所述,两分子IR780和一分子AIEgens的“工”字形反应产物显示出良好的光疗潜力。因此,用氨基、胍衍生物或苯硼酸衍生物对“工”字形化合物进行修饰可能使得到新分子具有针对幽门螺杆菌及其生物膜的潜在生物活性。本发明中,我们将R基(R=氨基、胍基或苯硼酸衍生物)功能化的IR780与AIEgens结合设计并合成了一类新型“工”字型光疗剂(T780T-R),其结构式如图(1)所示。具有靶向R基,T780T-R可以穿透生物膜并吸附在H.pylori表面。然后在低功率NIR激光照射(0.3W/cm2)下,T780T-R将光转化为热量和ROS,导致H.pylori的蛋白质变性和细胞膜破裂,通过有效穿透生物膜屏障和破坏生物膜,有效破坏预先形成的生物膜封闭的细菌。
本发明以R基为胍基提供的抗菌光敏剂T780T-Gu为例进行说明,其中T780T-Gu的合成路线图见附图1
本发明提供抗菌光敏剂T780T-Gu的制备方法,包括步骤如下:
在氮气保护下,将三氯氧磷(17.5mL,115mmol)逐滴加入到二氯甲烷/N,N-二甲基甲酰胺(40mL,1/1v/v)的搅拌溶液中,将所得溶液在冰浴下搅拌0.5h后,加入环己酮(7.6mL,50mmol)并在80℃下搅拌6h。将反应溶液倒入预冷的蒸馏水中置于4℃过夜,重结晶后抽滤,使用蒸馏水反复洗涤结晶物,干燥得到化合物1,为黄色固体(4.63g,26.8mmol,53.5%)。
在氮气保护下,将3-溴丙胺氢溴酸盐(2.74g,12.51mmol)加入到2,3,3-三甲基吲哚(2mL,12.5mmol)的搅拌溶液中,将所得溶液在120℃下搅拌10h后,将反应室温静置过夜。然后将所得残余物溶于少量二氯甲烷/甲醇(10/1v/v)中,并加入二氧六环/甲醇(5/1v/v)使产物重结晶,抽滤,使用二氧六环反复洗涤结晶物,干燥得到化合物2,为粉红色固体(0.85g,3.91mmol,31.28%)。
在氮气保护下,将化合物1(0.4mg,2.32mmol)和化合物2(1.12g,5.15mmol)加入到50mL正丁醇/甲苯(7/3v/v)的搅拌溶液中,将所得溶液在120℃下搅拌16h后,将反应混合物减压浓缩。然后将所得残余物溶于少量二氯甲烷/甲醇(10/1v/v)中,并加入大量正己烷使产物重结晶,抽滤,使用二氯甲烷反复洗涤结晶物,干燥得到化合物3,为金绿色固体(0.77g,1.34mmol,57.9%)。
在氮气保护下,将N,N-二-Boc-1H-吡唑-1-甲脒(4.33g,14.02mmol)加入到化合物3(1g,1.75mmol)的DMF(50mL)搅拌溶液中,将所得溶液在室温下搅拌72h后,将反应混合物减压浓缩。然后使用二氯甲烷/甲醇(100/1至40/1,V/V)通过柱色谱法(干法上样)纯化所得粗产物,得到化合物4,为绿色固体(0.69g,0.65mmol,37.32%)。
在氮气保护下,将1,2-二(4-羟基苯)-1,2-二苯乙烯(0.16g,0.43mmol)加入到化合物4(1g,0.95mmol)的DMF(50mL)搅拌溶液中,将所得溶液在室温下搅拌60h后,将反应混合物减压浓缩。然后使用二氯甲烷/甲醇(8/1,V/V)通过薄层色谱法分离法纯化所得粗产物,得到化合物5,为绿色固体(0.38g,0.15mmol,36.68%)。
在氮气保护下,将化合物5(0.45g,0.187mmol)溶解在10mL 4N HCl/1,4-二氧六环中,所得溶液在室温下搅拌过夜。减压蒸馏除多余溶剂后,将所得残渣溶于少量无水甲醇中,滴加大量乙醚使产物重结晶,抽滤,使用二氧六环/甲醇(3/1v/v)洗涤结晶物,真空干燥后得到目标化合物T780T-Gu,为绿色固体(0.297g,0.17mmol,91%)。
与现有技术相比,本发明的增益效果为:
本发明的抗菌光敏剂具有以下特点:(1)该光敏剂光热转换效率以及ROS生成能力高,抗光漂白能力强;(2)该光敏剂中的胍基基团使其对于细菌具有特异靶向性,生物兼容好;(3)该光敏剂的吸收波长处于近红外区(780nm),该波长的光具有更好的组织穿透能力;(4)该光敏剂在应用于抗菌治疗时可以避免细菌耐药性的发生。
附图说明
图1为本发明光敏剂的合成路线。
图2为本发明抗菌光敏剂(10μM)分别在DMSO、乙醇和水溶液中的紫外吸收图谱、荧光发射图谱和对应的透射电镜图。
图3为本发明光敏剂的热循环,热循环过程中紫外吸收光谱、颜色变化与ROS产生图。
图4为本发明抗菌剂对于浮游H.pylori ATCC 43504菌株的杀伤以及T780T-Gu定位在H.pylori的荧光共聚焦激光扫描显微镜(CLSM)成像图和T780T-Gu存在下,激光照射诱导胞内ROS生成的CLAM。
图5为本发明抗菌光敏剂的细胞毒性图。
图6为在本发明抗菌光敏剂存在下,激光照射抑制H.pylori ATCC 43504菌株形成生物膜的图像。
图7为本发明抗菌光敏剂对于H.pylori ATCC 43504菌株生物膜内细菌的杀伤CLSM图像。
图8为本发明抗菌光敏剂对于多重耐药H.pylori 7-18-4菌株生物膜内细菌的杀伤CLSM图像,以及对应细菌的透射电镜图像。
具体实施方式
下面结合附图和实例,对本发明抗菌光敏剂及其制备方法和应用的具体实现作进一步说明。
实施例1:
紫外可见吸收光谱、荧光吸收光谱和透射电子显微镜(TEM):分别配制10μMT780T-Gu的DMSO、乙醇和水溶液,进行300nm-900nm的紫外可见光谱扫描并进行荧光测试。对于TEM:将T780T-Gu溶液的稀释悬浮液沉积在碳涂层铜网格上并室温干燥48h,使用TEM观察样品的形貌,结果如图2。
实施例2:
光稳定性与ROS产生:配制10μM T780T-Gu的DMSO溶液,用0.3W/cm2(808nm)激光照射10分钟,然后停止照射冷却至室温,并记录其紫外吸收光谱和颜色,重复此步骤四次,在加热和冷却过程中每隔30秒记录一次溶液的温度。用ABDA法测定ROS的产生,用0.3W/cm2(808nm)激光照射15分钟,每隔一分钟扫描378nm处紫外吸收峰值。结果如图3。
实施例3:
浮游H.pylori ATCC 43504的杀伤及TEM样品制备:将幽门螺杆菌转移到布鲁氏菌肉汤(含有4%胎牛血清和幽门螺杆菌选择性抗生素)中并稀释至MCF=0.5。将细菌溶液加入到96孔板中(每孔100μL),每孔加入10μM T780T-Gu并孵育2小时。然后,将细菌暴露于激光照射(808nm,0.3W/cm2)10分钟。孵育2小时,将菌液系列稀释后,采用铺板法置于哥伦比亚琼脂平板上,培养3-5天,观察菌落数。对于透射电镜,将经历杀伤实验的浮游幽门螺杆菌溶液在2.5%戊二醛中4℃下固定4小时。然后将上述菌液离心(5000rpm,5min),分别用30%、50%、70%、80%、90%梯度乙醇处理10min,最后100%乙醇处理15min脱水。最后,将所得样品滴在碳涂层铜网格上,使用TEM观察幽门螺杆菌的形态,结果如图4A。
实施例4:
荧光共聚焦激光扫描显微镜成像:将幽门螺杆菌转移到Dulbecco磷酸盐缓冲盐水(DPBS)中并稀释至0.5麦氏浊度(MCF=0.5)。将细菌与10μM T780T-Gu在37℃下孵育2小时。用DPBS洗涤后,通过CLSM对细菌进行成像,结果如图4B。
实施例5:
细菌内ROS的测定:使用二氯荧光素二乙酸酯(H2DCF-DA)测量细胞内ROS。将幽门螺杆菌转移到布鲁氏菌肉汤(含有4%胎牛血清和幽门螺杆菌选择性抗生素)(MCF=0.5)中,并与10μM的H2DCF-DA一起孵育30分钟。然后将负载H2DCF-DA的细菌与10μMT780T-Gu在37℃下孵育2小时。之后暴露于激光照射(808nm,0.3W/cm2)10分钟,然后在37℃下再孵育2小时。用DPBS洗涤后,通过CLSM对细菌进行成像,结果如图4C。
实施例6:
将HUVEC细胞以每孔20000个的密度接种于96孔板中,培养24h。将含有T780T-Gu的母液分别用培养基稀释成不同的浓度(0.195、0.391、0781、1.563、3.125、6.25、12.5、25μM)。将培养板中的培养基吸出,加入不同浓度的T780T-Gu,分别在24及48小时后,加入MTT溶液(每孔20μL,5mg/mL),孵育4小时。吸出MTT溶液与培养基的混合液,每孔加入
150μL DMSO溶液,摇床震荡以充分溶解形成的甲瓒晶体物质。使用酶标仪记录570nm处的紫外吸收值,并使用以下公式计算细胞得生长活力:活力(%)=(实验组平均吸光度值/对照组平均吸光度值)*100%,得到细胞毒性数据,如图5。
实施例7:
幽门螺杆菌生物膜的培养、结晶紫染色和生物量定量:将H.pylori转移到布鲁氏菌肉汤(含有H.pylori选择性抗生素)并稀释至MCF=1。将溶液添加到96孔板或35mm共聚焦培养皿中,并在37℃下孵育。48小时后,将布鲁氏菌肉汤培养基更换为新鲜的布鲁氏菌肉汤,使生物膜再生长24小时。用结晶紫对幽门螺杆菌生物膜染色时,首先,在生物膜中加入4℃预冷的甲醇,固定生物膜10分钟。然后,将1%结晶紫(100μL)添加到幽门螺杆菌生物膜中。15分钟后,去除染料并用PBS漂洗3次。漂洗后,在染色后的生物膜中加入95%乙醇(100μL),用酶标仪测定570nm处的吸光度。
实施例8:
T780T-Gu抑制幽门螺杆菌生物膜形成的活性:将H.pylori转移到布鲁氏菌肉汤(含有H.pylori选择性抗生素)并稀释至MCF=1。在溶液中添加不同浓度的T780T-Gu(1μM、2μM、4μM、6μM、8μM、10μM),孵育2小时后,将幽门螺杆菌暴露于激光照射(0.3W/cm2,10分钟)。在72小时后测量生物膜生物量,结果如图6。
实施例9:
抗H.pylori ATCC 43504生物膜活性和生物膜中活/死H.pylori的SYTO 9/PI染色:预先在共聚焦培养皿上制备H.pylori ATCC 43504生物膜。对于抗生物膜测定,幽门螺杆菌生物膜与10μM T780T-Gu预孵育2小时,然后进行激光照射(808nm,0.3W/cm2)10分钟。之后,将细胞进一步孵育4小时。将细胞用5μM SYTO 9和30μM PI在0.85%NaCl中染色15分钟,并使用CLSM观察,结果如图7。
实施例10:
多药耐受H.pylori 7-18-4的E-test检测及T780T-Gu在抗多药耐受H.pylori 7-18-4生物膜中的活性:将幽门螺杆菌转移到布鲁氏菌肉汤(含有4%胎牛血清和幽门螺杆菌选择性抗生素)中并稀释至MCF=0.5。将100μL菌悬液涂在哥伦比亚琼脂平板上。然后,当琼脂平板完全干燥时,用镊子将E-test试纸放在平板表面。在微氧培养条件下培养72h后,观察细菌的耐药性。对于抗多重耐药H.pylori 7-18-4生物膜的活性,预先在共聚焦培养皿上制备H.pylori 7-18-4生物膜,将生物膜与10μM T780T-Gu预孵育2小时,然后进行激光照射(808nm,0.3W/cm2)10分钟。之后,将细胞进一步孵育4小时。将生物膜用5μM SYTO 9和30μMPI在0.85%NaCl中染色15分钟,并使用CLSM观察,结果如图8。
Claims (2)
1.细菌靶向光敏剂,其特征在于,光敏剂由两分子预修饰胍基基团的IR780与一分子TPE基团构成,其结构式如(1)所示:
2.如权利要求1所述的细菌靶向光敏剂的制备方法,其特征在于,制备路线如(2)所示:
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