CN107440795B - 一种双波长激励的反馈式光热治疗仪 - Google Patents
一种双波长激励的反馈式光热治疗仪 Download PDFInfo
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Abstract
一种双波长激励的反馈式光热治疗仪，它涉及光热治疗仪，它是要解决现有的激光光热治疗仪损害正常细胞、缺乏无创的温度反馈功能、治疗周期长的技术问题，该光热治疗仪包括双通道信号发生器、制热激光器、制冷激光器、光纤传输系统、上转换热疗探针、光谱仪和计算机；其中光纤传输系统包括由光纤依次连接的耦合器、环形器和聚焦镜；两束不同波长的脉冲激光分别用于加热和制冷，探针的上转换荧光信号解析探针温度，实现光疗过程中的温度反馈，并且进一步根据反馈的温度来调节输入激光参数，实现在线干预。本发明的治疗仪操作简单，可抑制过热现象、缩短治疗周期。
Description
技术领域
本发明涉及光热治疗仪，属于光学与医学的交叉领域。
背景技术
目前癌症治疗有三种主流方法：手术疗法，放射性疗法，化学疗法。其中手术疗法具有操作复杂、风险大、对人体创伤大、降低人体免疫力、以及容易出现并发症等缺陷；放射性疗法和化学疗法都会给患者带来了很大的副作用：这些方法在扼制癌细胞发展的同时，对正常细胞也产生巨大的伤害。
高温热疗通过提高体内温度杀死肿瘤，是一种低成本、易操作、副作用小的癌症治疗方法。一方面，高热可以抑制脱氧核糖核酸、核糖核酸及蛋白质的合成，进而促进肿瘤细胞死亡；另一方面，高热还可以损害细胞膜的正常功能，使细胞膜的通透性发生改变，引起蛋白外溢、核染色质结构发生改变，导致癌细胞的死亡。并且当温度达到40摄氏度以上，癌细胞会变得适于传统的化疗和放疗，所以癌症热疗法可以明显增加常规治疗手段对肿瘤的局部空置率、改善远期生存，具有其它治疗手段无法比拟的作用。
以激光进行高温治疗的方法被称为激光热疗，目前已成为肿瘤热疗的一种新的有效手段。现有的激光光热治疗仪通常采用稳态激光长时间照射以维持肿瘤组织处于较高温度，这种常规方案具有以下三个主要缺陷：一是容易引起病变组织周围健康细胞的过热效应，导致在癌症的光热治疗过程中，高温杀死肿瘤组织的同时也损害了正常细胞；二是在激光热疗过程中缺乏无创、便捷的温度反馈机制。由于温度是癌症热疗过程的关键治疗参数，所以实时监测光疗探针及周围组织的温度十分关键，但是当前的常规方法是将多个温度传感探头植入人体内不同位置进行监测，大大增加了热疗系统的复杂度和对人体的伤害；三是为保证较好的疗效，激光照射时间通常为60-120分钟，相对较长的治疗时间不利于某些特殊情况患者，如不能长时间保持身体姿态不变的患者。
针对以上常规的癌症激光热疗方法的缺陷，本发明提供了一种能够有效抑制过热效应、缩短疗程、且具备无创温度反馈功能的癌症热疗仪，该热疗仪结构紧凑、操作便捷，具有广阔的应用前景。
发明内容
本发明是要解决现有的激光光热治疗仪损害正常细胞、缺乏无创的温度反馈功能、治疗周期长的技术问题，提供一种双波长激励的反馈式光热治疗仪。
本发明的双波长激励的反馈式光热治疗仪结构如图1所示，包括双通道信号发生器1、制热激光器2、制冷激光器3、光纤传输系统4、上转换热疗探针5、光谱仪6和计算机7；
其中光纤传输系统4包括由光纤依次连接的耦合器4-1、环形器4-2和聚焦镜4-3；
制热激光器2和制冷激光器3并联后一端与双通道信号发生器1的输出端连接，另一端与光纤传输系统4的耦合器4-1连接，光纤传输系统4的聚焦镜4-3发出的激光照射在上转换热疗探针5上；
光纤传输系统4的环形器4-2的另一输出端与光谱仪6连接，光谱仪6的信号输出端与计算机7的输入端连接，计算机7的输出端与双通道信号发生器1的输入端连接。
进一步地，制热激光器2输出的激光波长为940nm；制冷激光器3输出的激光波长为1020nm；两束激光的频率范围为：0.1～5Hz，功率密度范围为：0.2～1W/cm2。
进一步地，上转换热疗探针5是单分散的Er3+/Yb3+:LiLuF4@LiYF4异质结构纳米晶；
进一步地，上转换热疗探针5的制备方法如下：
一、Er3+/Yb3+:LiLuF4纳米晶的制备：在高纯氩气的保护下，按ErCl3、YbCl3、LuCl3、LiOH、NH4F的摩尔比为1：(10～30)：(19～39)：(100～150)：(200～300)的比例，称取ErCl3、YbCl3、LuCl3、LiOH和NH4F；先将ErCl3、YbCl3、LuCl3加入油酸与十八烯的混合溶液中升温至140～160℃保持15～45分钟，形成稀土油酸盐；待降至室温后再加入LiOH和NH4F的甲醇溶液，升温至140～160℃保温排出溶液中的甲醇和水后，再升温至300～320℃并保温2小时，得到纳米晶产物经离心清洗后分散至环己烷中，即得到Er3+/Yb3+:LiLuF4纳米晶的环己烷分散液；
二、Er3+/Yb3+:LiLuF4@LiYF4核壳结构纳米晶的制备：按YCl3、LiOH与NH4F的摩尔比为1：(2～4)：(4～6)的比例称取YCl3、LiOH和NH4F；首先，把YCl3加入OA和ODE的混合溶液中升温至140～160℃保持15～45分钟，形成稀土油酸盐；待降至室温后再加入LiOH和NH4F的甲醇溶液，升温至140～160℃保温排出溶液中的甲醇和水后，降至室温作为壳层原液待用；然后，将步骤一制备的Er3+/Yb3+:LiLuF4纳米晶的环己烷分散液加入三口烧瓶中，加入油酸和十八烯混合均匀后升温至140～160℃保持15～45分钟，之后将溶液升温至300～310℃并保持恒温；最后，取壳层原液分次注射进入含有Er3+/Yb3+:LiLuF4纳米晶的烧瓶，注射完成后反应液降至室温，经离心清洗后分散至环己烷中，即获得Er3+/Yb3+:LiLuF4@LiYF4核壳结构纳米晶；
三、表面修饰叶酸：在氮气的保护下，将叶酸、4-二甲基氨基吡啶、上转换纳米晶Er3+/Yb3+:LiLuF4@LiYF4加入无水二甲基甲酰胺中搅拌均匀，其中叶酸、4-二甲基氨基吡啶与上转换纳米晶Er3+/Yb3+:LiLuF4@LiYF4的质量比为1：1：(1～2)；再加入二环己基碳二亚胺和三乙胺搅拌20～30小时，其中二环己基碳二亚胺与叶酸的质量比为(1.4～1.8)：1，三乙胺体积与叶酸的用量比为(2～3)微升：1毫克；添加过量的二乙醚沉淀样品，离心清洗后分散至去离子水中，获得外表面修饰叶酸的Er3+/Yb3+:LiLuF4@LiYF4纳米晶，即上转换热疗探针。
该热疗仪的原理示意图如图2所示，其具体工作原理如下：分别使用940nm激光器和1020nm激光器作为治疗仪的加热及制冷光源。探针中作为吸收中心的Yb离子的最强吸收位置在980nm左右，当采用940nm激光作加热光源，由于此时入射光子的能量略高于Yb离子的能级间距，Yb在吸收入射激光能量的同时，超出的能量将转化为基质的晶格振动，即转化为热能实现激光加热；另一方面，当采用1020纳米激光入射时，光子能量略低于Yb的能级间距，吸收仍可发生，只是该过程需要基质声子的辅助，即从基质吸收能量以桥接入射光子与吸收中心的能量失配，所以最终会导致上转换材料温度降低，实现制冷。同时由于在上述两种激光波长的照射下，Yb离子均可以吸收能量，进而将能量传递给发光稀土离子Er3+，故在两种波长激励下，探针均可产生上转换荧光信号。
本发明所设计的治疗仪中，为了避免加热与制冷的相互干扰，通过入射激光的脉冲输入方式实现探针升温与降温的交替变化。波长为940nm的制热激光器、波长为1020nm的制冷激光器发出的脉冲激光导入光纤耦合器，经环形器后到达光纤出射端，通过光纤端的聚焦镜汇聚至人体内的光疗探针上。两束激光的频率范围为：0.5～5Hz，功率密度范围为：0.2～1W/cm2。940nm和1020nm激光不仅用于加热和制冷，还同时激发探针产生上转换荧光，经聚焦镜耦合反向进入光纤后，再通过环形器的另一输出端进入光谱仪，利用计算机进行信号处理，解析出探针的温度信息作为光热治疗的反馈参数，通过计算机控制双通道信号发生器的输出，用于调节两路激光的输入参数，即波形、功率、频率和占空比。
本发明采用热分解法制备稀土上转换荧光材料作为光疗探针，其中上转换荧光过程为：稀土离子吸收多个低能量的近红外光子后，跃迁至较高能级发射紫外/可见光子。热疗探针为单分散的外表面连接叶酸的Er3+/Yb3+:LiLuF4@LiYF4纳米晶，其中敏化离子Yb3+具有较强近红外吸收能力，并且通过较高的Yb离子掺杂浓度来提高探针的光热转换能力；发光离子Er3+可以与Yb离子实现高效的能量传递；基质LiLuF4比常用的上转换材料NaYF4更适于稀土离子掺杂产生高效的上转换荧光，为了降低成本且进一步提高探针的上转换效率，在Er3+/Yb3+:LiLuF4的表面包裹异质结构的LiYF4保护层形成核壳结构纳米晶。为了实现探针在人体内与肿瘤组织的连接，在探针表面进一步连接叶酸，用于靶向癌细胞。
本发明基于探针的上转换荧光信号反馈探针温度信息。温度反馈功能通过荧光强度比技术实现，其原理如图3所示：两个临近的热耦合能级(1和2)上粒子数布居遵循玻尔兹曼分布，故各自向下跃迁产生的荧光强度比值R与温度T之间满足：
其中I2和I1分别为两热耦合能级发射的荧光强度值，C为由材料性质决定的常数，ΔE为热耦合能级的间距，k为玻尔兹曼常数。
对于本发明中所采用的Er3+离子发光系统，其绿光能级2H11/2和4S3/2上的布居粒子数满足玻尔兹曼分布，因而可以通过测量不同温度下探针的上转换荧光的光谱，求得荧光强度比值，利用公式(1)计算探针的温度，用于温度反馈。
本发明的双波长激励的反馈式光热治疗仪的有益效果如下：
将稀土上转换材料作为探针用于癌症热疗具有独特的优势：一是利用近红外激光作为激励源，可以显著增大激光在人体内的透射深度；二是近红外激光在人体组织当中产生的背景荧光很低，可以保证较高的上转换荧光信噪比；三是稀土掺杂纳米荧光材料的生物毒性低；四是上转换材料的稳定性好，不易出现光漂白；五是这种纳米尺度探针的空间分辨率高。
本发明：采用脉冲激光在时域上分离加热与制冷作用，并使用较高的激发光功率实现探针温度的剧烈变化，这种温度的剧烈变化有利于在短时间内杀死癌症细胞，缩短疗程。
本发明：在采用脉冲激光且引入制冷机制后，相对常规稳态激光照射的癌症光热治疗技术，激光对探针周围健康组织的过热效应得到有效的抑制，可以降低激光热疗的副作用。
本发明：采用纯LiYF4保护层包裹Er/Yb:LiLuF4，一方面可以隔绝稀土发光中心向纳米晶表面缺陷的能量传递，提高上转换荧光效率；另一方面，利用低成本的YCl3原料替代昂贵的LuCl3能够极大的降低探针的制备成本。
本发明：基于探针的上转换信号，通过荧光强度比技术监测温度，实现热疗过程的无损温度反馈，进而调整输入激光参数(两束激光各自的输入功率、波形、频率等)，实现治疗的在线干预，优化疗效。
附图说明
图1是本发明双波长激励的反馈式光热治疗仪的结构示意图。其中1为双通道信号发生器、2为制热激光器、3为制冷激光器、4为光纤传输系统、5为上转换热疗探针、6为光谱仪6，7为计算机；4-1为耦合器、4-2为环形器、4-3为聚焦镜；
图2是本发明双波长激励的反馈式光热治疗仪中的加热及制冷的原理示意图；
图3是本发明双波长激励的反馈式光热治疗仪的探针的温度反馈工作原理图；
图4是实施例1中制备的Er3+/Yb3+:LiLuF4纳米晶的TEM照片；
图5是实施例1中制备的经过表面修饰的上转换热疗探针Er3+/Yb3+:LiLuF4@LiYF4的TEM照片；
图6是实施例1中用双波长激励的反馈式光热治疗仪的使用示意图。其中8为肿瘤组织，9为正常组织；
图7是实施例1中双波长激励的反馈式光热治疗仪的脉冲输入方式示意图；
图8是实施例1中双波长激励的反馈式光热治疗仪在不同激光输入方式下，探针的温度随时间变化规律图；
图9是实施例1中双波长激励的反馈式光热治疗仪在不同激光输入方式下，临近探针的正常组织的温度随时间的变化规律图。
具体实施方式
用以下实施例验证本发明的有益效果：
实施例1：本实施例的双波长激励的反馈式光热治疗仪由双通道信号发生器1、制热激光器2、制冷激光器3、光纤传输系统4、上转换热疗探针5、光谱仪6和计算机7组成；其中光纤传输系统4由耦合器4-1、环形器4-2和聚焦镜4-3组成并且由光纤依次连接；
制热激光器2和制冷激光器3并联后一端与双通道信号发生器1的输出端连接，另一端与光纤传输系统4的耦合器4-1连接，光纤传输系统4的聚焦镜4-3发生的激光照射在上转换热疗探针5上；
光纤传输系统4的环形器4-2与光谱仪6连接，光谱仪6的信号输出端与计算机7的输入端连接，计算机8的输出端与双通道信号发生器1的输入端连接；
制热激光器2输出的激光波长为940nm；制冷激光器3输出的激光波长为1020nm；
上转换热疗探针5是单分散的外表面修饰叶酸的Er3+/Yb3+:LiLuF4@LiYF4异质结构纳米晶；其制备过程如下：
一、Er3+/Yb3+:LiLuF4纳米晶的制备：为了获得适用于生物应用的形貌均一、单分散的上转换纳米晶，采用热分解法合成稀土掺杂LiLuF4纳米晶。具体步骤如下：首先，将摩尔比为2：18：80，总量为1mmol的ErCl3、YbCl3、LuCl3加入6ml的油酸和15ml的十八烯中升温至160℃形成稀土油酸盐，待降至室温后再加入含有2.5mmol的LiOH和4mmol的NH4F的10ml甲醇溶液，经过150℃保温30分钟排出溶液中的甲醇和水后，混合液被加热至300～320℃并保温2小时，纳米晶产物经离心清洗后分散至5ml环己烷中；为了避免产物氧化，反应全程在高纯氩气的保护下进行；步骤一制备的Er3+/Yb3+:LiLuF4纳米晶的TEM照片如图4所示，图中标尺为50纳米；从图4可以看出，Er3+/Yb3+:LiLuF4纳米晶为单分散的菱形颗粒；
二、Er3+/Yb3+:LiLuF4@LiYF4核壳结构纳米晶的制备：一方面，将1mmol的YCl3加入6ml的油酸和15ml的十八烯中升温至160℃形成稀土油酸盐，待降至室温后再加入含有2.5mmol的LiOH和4mmol的NH4F的10ml甲醇溶液，经过150℃保温排出溶液中的甲醇和水后，降至室温作为壳层原液待用；另一方面，将1ml之前制备的Er3+/Yb3+:LiLuF4纳米晶的环己烷分散液加入50ml三口烧瓶，再加入6ml的油酸和15ml的十八烯中升温至150℃保持30分钟，之后将溶液升温至300℃保持恒温；最后，取4ml壳层原液分20次注射进入含有Er3+/Yb3+:LiLuF4纳米晶的烧瓶，每次注射间隔3分钟。注射完成后反应液降至室温，经离心清洗后，获得异质结构Er3+/Yb3+:LiLuF4@LiYF4，即为上转换纳米晶；
三、表面修饰叶酸：在氮气的保护下，将10mg叶酸、10mg的4-二甲基氨基吡啶、20mg上转换纳米晶加入2ml无水二甲基甲酰胺中，加入16mg二环己基碳二亚胺和20μl三乙胺搅拌24小时；通过添加过量的二乙醚沉淀样品，离心清洗后分散至去离子水中，获得表面修饰叶酸的上转换纳米晶，即为上转换热疗探针；上转换热疗探针的透射电镜图如图5所示，其中标尺为20纳米。从图5可以看出，在单分散的菱形颗粒外表面有包覆层；
利用模拟的人体组织及HeLa细胞测试本实施例的双波长激励的反馈式光热治疗仪的使用效果。将HeLa细胞植入培养皿，在37℃和浓度为5％的CO2环境下培育24小时，再将HeLa细胞与上转换热疗探针的水分散液一同加入RPMI-1640培养基中培育4小时，经PBS清洗后，注射进入2cm×2cm×5mm猪皮组织中。
将制热激光器2、制冷激光器3发出的激光导入光纤传输系统4中，具体过程如下：首先将两束激光分别通过耦合器4-1的两路输入端口导入光纤中，激光信号经过环形器4-2后，由聚焦镜4-3汇聚光斑至上转换热疗探针5位置上；
探针5在激光的照射下产生上转换荧光信号，该荧光信号通过聚焦镜4-3反向耦合进入光纤，再由环形器4-2的另一出口出射，进入光谱仪6，光谱仪6测试的信息通过计算机7利用公式(1)解析上转换热疗探针5的温度值；
图6是本实施例1中双波长激励的反馈式光热治疗仪的使用示意图。其中8为肿瘤组织，9为正常组织；
设置双通道信号发生器1的脉冲输入方式参数如图7所示，其中两束激光均为周期为5秒的方波输入，940nm激光的功率密度为0.4W/cm2，波形占空比为60％；1020nm激光的功率密度为0.5W/cm2，波形占空比为40％。调整信号发生器的输入使得两束激光的作用在时域上分开，实现940nm激光与1020nm激光的交替输入，用于双波长激光热疗。
本实施例的双波长激励的反馈式光热治疗仪的激光中心点温度变化与常规方法的进行对比，其中双波长激励的反馈式光热治疗仪是高功率的双脉冲照射，常规方法是低功率的连续波照射。两种方法不同激光输入方式下，探针的温度随时间变化情况如图8所示，由图8可以看出，相对采用连续激光照射的方法，在平均温度相等的前提下，高功率的双脉冲激励方式能够实现光疗探针温度的剧烈变化，表明双波长激励的反馈式光热治疗仪可以缩短治疗周期。
本实施例的双波长激励的反馈式光热治疗仪中，距离上转换热疗探针1.5mm处组织的温度变化与常规的低功率稳态激光连续长时间照射的进行对比，得到的距上转换热疗探针1.5mm处正常组织的温度随时间变化关系图如图9所示。从图9可以看出，两种方式下光疗探针临近位置的温度变化随激光激励方式差异明显。当采用低功率的连续激光照射时，周围组织持续升温，2分钟后即可达42摄氏度，在长时间低功率激光热疗的作用下，将导致健康组织的受损。而另一方面，采用本实施例的高功率的双脉冲方式照射，由于引入了制冷机制，周围组织的升温得到了明显的抑制，2分钟后该位置的温度仍保持在40摄氏度左右。由于双脉冲激励方式的治疗周期较短，上述结果表明双波长激励的反馈式光热治疗仪有利于保护光热治疗过程中健康组织不受高温的损害。
Claims (5)
1.一种双波长激励的反馈式光热治疗仪，其特征在于它包括双通道信号发生器(1)、制热激光器(2)、制冷激光器(3)、光纤传输系统(4)、上转换热疗探针(5)、光谱仪(6)和计算机(7)；
其中光纤传输系统(4)包括由光纤依次连接的耦合器(4-1)、环形器(4-2)和聚焦镜(4-3)；
制热激光器(2)和制冷激光器(3)并联后一端与双通道信号发生器(1)的输出端连接，另一端与光纤传输系统(4)的耦合器(4-1)连接，光纤传输系统(4)的聚焦镜(4-3)发出的激光照射在上转换热疗探针(5)上；
光纤传输系统(4)的环形器(4-2)与光谱仪(6)连接，光谱仪(6)的信号输出端与计算机(7)的输入端连接，计算机(7)的输出端与双通道信号发生器(1)的输入端连接；
其中上转换热疗探针(5)的制备方法如下：
一、Er3+/Yb3+:LiLuF4纳米晶的制备：在高纯氩气的保护下，按ErCl3、YbCl3、LuCl3、LiOH、NH4F的摩尔比为1：(9～10)：(39～40)：(100～150)：(200～300)的比例，称取ErCl3、YbCl3、LuCl3、LiOH和NH4F；先将ErCl3、YbCl3、LuCl3加入油酸与十八烯的混合溶液中升温至140～160℃保持15～45分钟，形成稀土油酸盐；待降至室温后再加入LiOH和NH4F的甲醇溶液，升温至140～160℃保温排出溶液中的甲醇和水后，再升温至300～320℃并保温2小时，得到的纳米晶产物经离心清洗后分散至环己烷中，即得到Er3+/Yb3+:LiLuF4纳米晶的环己烷分散液；
二、Er3+/Yb3+:LiLuF4@LiYF4核壳结构纳米晶的制备：按YCl3、LiOH与NH4F的摩尔比为1：(2～4)：(4～6)的比例称取YCl3、LiOH和NH4F；首先，把YCl3加入油酸和十八烯的混合溶液中升温至140～160℃保持15～45分钟，形成稀土油酸盐；待降至室温后再加入LiOH和NH4F的甲醇溶液，升温至140～160℃保温排出溶液中的甲醇和水后，降至室温作为壳层原液待用；然后，将步骤一制备的Er3+/Yb3+:LiLuF4纳米晶的环己烷分散液加入三口烧瓶中，加入油酸和十八烯混合均匀后升温至140～160℃保持15～45分钟，之后将溶液升温至300～310℃并保持恒温；最后，取壳层原液分次注射进入含有Er3+/Yb3+:LiLuF4纳米晶的烧瓶，注射完成后反应液降至室温，得到的纳米晶经离心清洗后，即获得Er3+/Yb3+:LiLuF4@LiYF4核壳结构纳米晶；
三、表面修饰叶酸：在氮气的保护下，将叶酸、4-二甲基氨基吡啶、上转换纳米晶Er3+/Yb3+:LiLuF4@LiYF4加入无水二甲基甲酰胺中搅拌均匀，其中叶酸、4-二甲基氨基吡啶与上转换纳米晶Er3+/Yb3+:LiLuF4@LiYF4的质量比为1：1：(1～2)；再加入二环己基碳二亚胺和三乙胺搅拌20～30小时，其中二环己基碳二亚胺与叶酸的质量比为(1.4～1.8)：1，三乙胺体积与叶酸的用量比为(2～3)微升：1毫克；添加过量的二乙醚沉淀样品，离心清洗后分散至去离子水中，获得外表面修饰叶酸的Er3+/Yb3+:LiLuF4@LiYF4纳米晶，即上转换热疗探针。
2.根据权利要求1所述的一种双波长激励的反馈式光热治疗仪，其特征在于制热激光器(2)输出的激光波长为940nm。
3.根据权利要求1或2所述的一种双波长激励的反馈式光热治疗仪，其特征在于制冷激光器(3)输出的激光波长为1020nm。
4.根据权利要求1或2所述的一种双波长激励的反馈式光热治疗仪，其特征在于两束激光的频率范围为：0.1～5Hz，功率密度范围为：0.2～1W/cm2。
5.根据权利要求1或2所述的一种双波长激励的反馈式光热治疗仪，其特征在于，通过双通道信号发生器的调节，实现两束激光为交替输入的脉冲方波。
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