This research is funded by an operating grant from the Canadian Institutes of Health Research (CIHR) to C.A. and D.A.J. The authors acknowledge the Canadian Foundation for Innovation and Princess Margaret Cancer Foundation for funding the STTARR research facility that enables the imaging and therapy research components of this work.

1.脂质体制备<ol><li>溶解脂质1,2- Dipalmitoyl- SN -glycero -3-磷酸胆碱（DPPC），1-硬脂酰-2-羟基- SN -glycero -3-磷脂酰胆碱（MSPC或S-溶血PC）和N - （羰基甲氧基聚乙二醇2000）-1,2- distearoyl- SN -glycero -3-磷酸（MPEG 2000 -DSPE）的氯仿。例如，对于准备以10ml HTLC的，称出314.4毫克DPPC，39.4毫克MSPC，和83.9毫克的mPEG 2000 -DSPE成琥珀色玻璃小瓶中。然后，溶解脂质在2毫升氯仿和加热小瓶30秒在60℃的水浴中。 </li><li>除去使用旋转蒸发器将氯仿。放置在真空高压O / N所产生的脂质薄膜。 </li><li> 10 ml的HTLC的，水合用5ml 0.1N Tris缓冲液（pH7.4）中1小时的脂质膜。与此同时，称量出162.4毫克1,2- dipalmitoyl- SN -glycero -3-磷酸甘油（DPPG）类脂和100mg顺铂粉末和水合物瓦特第i 5 ml的0.1N含30％乙醇（pH7.4）中1小时的Tris缓冲液中。 </li><li>对于钆HTLC制剂，称出DPPG脂质和10mg CDDP粉末的相同的量，然后用2.5毫升的Gd-HP-DO3A溶液（279.3毫克/毫升）加2.5含30％乙醇毫升0.1N Tris缓冲液水合（ pH7.4）中1小时。期间水合，该混合物必须保持在琥珀色小瓶中，并放置在热板上在70℃下在恒定搅拌下和涡旋每10分钟。 </li><li>结合脂质混合物和脂质药物混合物中，并再水合1小时。涡每次15-20分钟。 </li><li>组装10毫升挤出机的两个堆栈为200nm的聚碳酸酯过滤器。挤出机的thermobarrel连接到循环水浴在70℃，挤出机连接到一个压缩氮气罐。 </li><li>立即水合后，将混合物转移到挤出机腔室。打开氮气流（设定压力至200psi）通过膜挤出脂质体。收集liposomES在50ml管。保持管中的热水浴（70℃）在任何时候都在挤出过程。重复此过程5次。 </li><li>拆卸挤出机改为两叠100 nm的聚碳酸酯过滤器。重新组装挤出机压力设为400磅。重复步骤5，除了挤出脂质体10次。从最终挤出的样品收集到15毫升管。 </li><li>降温的脂质体至RT，并离心机在1000×g离心3分钟以沉淀不溶CDDP。 </li><li>透析脂质体O / N对0.9％的盐水中透析管与15000分子量截留（MWCO）在无菌条件下。 </li><li>稀释10微升在990微升蒸馏水中的脂质体。使用动态光散射来测量HTLC和钆HTLC的尺寸分布。进行3次测量各10次。 </li><li>稀释40微升3,960微升蒸馏水脂质体。使铂和钆5-6标准溶液在0.1至20微克/毫升的浓度范围。使用电感耦合等离子体原子发射光谱仪（ICP-AES）在3个不同的波长测定铂和钆的浓度。进行3次测量在每个波长平均结果。 </li><li>确定凝胶来使用差示扫描热量计（DSC）的HTLC脂质体制剂的液晶相转变温度（T M）。负载5-10毫克HTLC成DSC盘中，并使用空盘作为参比。使用5℃/分钟的扫描速率斜坡上升的温度从0℃至60℃。 </li><li>包裹脂质体中铝箔防止光曝光，并储存于4℃。 </li></ol> 2.从脂质体体外释放 <ol><li>准备离心柱。 <ol><li>孵育在400ml 0.9％的盐水50个克凝胶过滤珠在RT 3-4小时。卷起一块玻璃棉，湿用0.9％的盐水，将其放入1毫升注射器的尖端。按玻璃棉以填充约注射器的0.05-0.1毫升。使用玻璃吸管逐渐添加约1ml的凝胶过滤介质进入注射器。 </li><li>将注射器入15ml试管并离心在1000×g离心3分钟。取出旋转柱，并放入15毫升管。 </li></ol></li><li>样品400微升HTLC或Gd-HTLC成8 1英钱玻璃小瓶中并在任一37℃或42℃孵育在恒温水浴中。取出一个小瓶在每个时间点（即，5，15，30和60分钟），并立即将其置于冰上。 </li><li>加入100微升的0.9％盐水到准备好的离心柱，然后孵育（对照）前添加100μl的HTLC或Gd-HTLC的或孵化的水浴到离心柱后。离心在1000×g离心3分钟。从管中取出注射器和稀释在管对于ICP-AES分析的溶液。 </li></ol> 3.颈椎植入皮下异种瘤<ol><li>所有的动物研究，根据批准的大学健康网络的动物保健委员会（UHN）动物使用的协议进行的。 </li><li>请在生物安全柜（BSC）的所有动物研究。高压灭菌所有手术工具的任何手术前。每个手术后，擦拭用70％乙醇的外科手术工具和消毒用热珠灭菌一次。 </li><li>对于安乐死，将动物放在一个绝缘的CO 2室中，然后执行颈椎脱位。 </li><li>对于使用100％的氧气全身麻醉，用5％异氟烷诱导和维持2％。为了确保麻醉深度，施加压力钳的足垫掌面，观察动物反应。敷眼润滑剂，以防止干燥，而在麻醉下。 </li><li>对于已经经历手术的动物，放置在一个干净笼子的动物没有其他动物的公司。监视直到有足够的意识正在苏醒。 </li><li>文化在ME-180细胞中的α-极小必需培养基（α-MEM），补充有10％胎牛血清（FBS）和抗生素。 </li><li>收获细胞，并保持细胞的完全培养基接种前。计数使用血球细胞。 </li><li>每个接种供体小鼠承担肌肉注射（IM）ME-180（人宫颈癌细胞）肿瘤。 注：生长在即时通讯站点，以便确保良好的血管瘤的发展被选择。购买雌性SCID小鼠（6〜8周龄，约20克）从内部养殖基地。 <ol><li>麻醉雌性SCID小鼠和注射1×10 6个ME-180细胞进入后肢的腓肠肌用27G的针。 </li><li>使用卡尺，直到肿瘤达到9-12毫米，其最大尺寸测量肿瘤的大小。终止研究如果肿瘤已经超过12mm时，或者如果肿瘤块损害正常行为，行走，食物和水的摄入。 </li></ol></li><li>植入肿瘤块从供体小鼠到受体小鼠。 <ol><li>麻醉用5％异氟烷供体小鼠。使用麻醉下颈椎脱臼安乐死鼠标。取下供体小鼠供体肿瘤。切肿瘤为2-3毫米的3立方米的碎片。 </li><li>麻醉，用5％异氟烷接受者小鼠。注入0.5毫克100微升/毫升昔康皮下手术前。适用的碘手术擦洗溶液，然后70％的乙醇，最后碘溶液的剃光皮肤上。剃须的受体小鼠的左后肢。做一个切口处的皮肤的水平。将一个供体肿瘤块皮下通过切口。关闭使用1-2伤口剪辑（S）的切口。 </li><li>植入后取下夹子3-5天。皮下肿瘤需要使用基于激光的加热设置。 </li><li>允许肿瘤的加热处理之前生长2-3周。 </li></ol></li></ol> 4.设计，设备与供应一个形激光传输照明的体内暖气布莱和校准<ol><li>使用763纳米二极管激光器耦合到使用光纤的共形照明器实现组织加热。 <ol><li>照明提供了异种移植瘤的均匀表面的激光照射。它由含有三个零距离接触光积分腔室球与一个腔室中的中间（直径16mm）和图2的小室在任一侧（16毫米长和5毫米高反射材料30×20×17毫米的块的在直径）（ 图1）。 </li><li>为了让光从一个腔室传递到另一个，两个小直径5毫米的孔，​​小的外室和较大的中间室之间切割。光从激光输送到使用400微米的切断端光纤连接到通过一个600微米的直径的孔传递到腔室中的激光的外腔室。 </li><li>由于光的int性质eraction与腔室壁，光递送到的小室中的一个在空间上均匀化之后，通过内部端口进入较大的腔室，其中它是进一步空间上均匀化。光然后退出上中间室的直径10毫米的端口。 </li></ol></li><li>校准照明光传输相对于采用NIST的激光功率设置校准50毫米积分球测量的输出功率。 <ol><li>计算使用基于肿瘤的简化几何Monte Carlo模拟的光分布的肿瘤。产生在写入用市售的计算软件包和基座上的光传输蒙特卡洛模拟在Jacques等14的多层组织的标准代码的自定义算法的蒙特卡洛代码。 </li><li>模拟肿瘤为半球躺在平皮肤线的直径在皮肤表面和一个匹配的7毫米的平均肿瘤尺寸，他飞行的5毫米。表面通过随机发射的光子在整个暴露的半球和直接在球的中心模型均匀照明。 </li><li>使用的光学性能，包括吸收，μ 一个 = 0.025毫米-1，散射，μS = 10个毫米-1，各向异性因子克= 0.9和折射率，n = 1.4。在计算中使用百万光子。 </li><li>执行前，任何在体内的加热实验初始校准后没有进一步的修改，这些测量。 </li></ol></li></ol>使用自定义设计的激光室设置肿瘤的5形加热<ol><li>连接一个激光光纤到激光装置上，另一端向所述照射器的一端。 </li><li>麻醉，用5％异氟烷的动物。将一个27G的注射导管插入侧尾静脉鼠标。插入一个22克导管进入肿瘤的中心。将光纤温度公关OBE入空心导管监测温度的变化。 </li><li>覆盖与照明器（ 图2）整个肿瘤。首先将电源为0.8-1 W.打开激光，等待温度上升。通过手动调节0.1-0.8 W之间的激光功率维持肿瘤的在42℃的温度</li></ol> 6.温度分布的评估通过磁共振温度（MRT） <ol><li>测量通过质子谐振频率偏移（PRF移）MRT 7特斯拉临床前MR成象系统 15上结合的光纤温度探针测量使用基于激光的加热设置加热肿瘤的温度分布。 </li><li>以10秒的间隔用2D-FLASH脉冲序列（回波时间4.5毫秒;重复时间156.25毫秒）取得测温图像312 x 312μm的平面分辨率和光纤的水平2毫米层厚在一个片温度亲是。 </li></ol>代理发行的七，监测<ol><li>执行Ť1 -加权成像之前和钆HTLC给药后20分钟。 </li><li>植入物2的肿瘤使用在第3上述用于植入的方法的雌性SCID小鼠的，各有一个后肢，。预热的左后肢5分钟的肿瘤在42℃在注射前的Gd-HTLC的66.3毫克/公斤的Gd-HP-DO3A和1.4毫克/千克CDDP的剂量，然后加热该肿瘤为额外的20分钟后注射。期间加热处理进行注射。使用上的右后肢肿瘤作为未加热的控制。 </li><li>获取动态磁共振图像（36×20毫米的视场的，200×200微米的面内分辨率，2毫米切片厚度; MR序列协议）来监视的Gd-HP-DO3A释放在12秒的间隔在整个20分钟后期管理钆类水滑石的，刚开始30秒前注射。 </li><li>轮廓肿瘤（加热和不加热）和肌肉体积。每个轮廓体积内计算所有体素的平均MR信号。 </li></ol>

<ol>
	<li>Maeda, H., Wu, J., Sawa, T., Matsumura, Y., Hori, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+vascular+permeability+and+the+EPR+effect+in+macromolecular+therapeutics:+a+review.">Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review.</a> J Control Releas. 65 (1-2), 271-284 (2000).</li><li>Simard, P., Leroux, J. C., Allen, C., Meyer, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomes+for+Drug+Delivery.">Liposomes for Drug Delivery.</a> Nanoparticles for Pharmaceutical Application. , (2007).</li><li>O'Brien, M. E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+cardiotoxicity+and+comparable+efficacy+in+a+phase+III+trial+of+pegylated+liposomal+doxorubicin+HCl+(CAELYX+(TM)/Doxil+(R))+versus+conventional+doxorubicin+for+first-line+treatment+of+metastatic+breast+cancer.">Reduced cardiotoxicity and comparable efficacy in a phase III trial of pegylated liposomal doxorubicin HCl (CAELYX (TM)/Doxil (R)) versus conventional doxorubicin for first-line treatment of metastatic breast cancer.</a> Ann Onco. 15 (3), 440-449 (2004).</li><li>White, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+II+study+of+SPI-77+(sterically+stabilised+liposomal+cisplatin)+in+advanced+non-small-cell+lung+cancer.">Phase II study of SPI-77 (sterically stabilised liposomal cisplatin) in advanced non-small-cell lung cancer.</a> Br J Cancer. 95 (7), 822-828 (2006).</li><li>Laginha, K. M., Verwoert, S., Charrois, G. J. R., Allen, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+doxorubicin+levels+in+whole+tumor+and+tumor+nuclei+in+murine+breast+cancer+tumors.">Determination of doxorubicin levels in whole tumor and tumor nuclei in murine breast cancer tumors.</a> Clin Cancer Res. 11 (19), 6944-6949 (2005).</li><li>Baronzio, G. F., Hager, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Hyperthermia in cancer treatment: a primer. , (2006).</li><li>Landon, C. D., Park, J. Y., Needham, D., Dewhirst, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+Drug+Delivery+and+Hyperthermia:+The+Materials+Design+and+Preclinical+and+Clinical+Testing+of+Low+Temperature-Sensitive+Liposomes+Used+in+Combination+with+Mild+Hyperthermia+in+the+Treatment+of+Local+Cancer.">Nanoscale Drug Delivery and Hyperthermia: The Materials Design and Preclinical and Clinical Testing of Low Temperature-Sensitive Liposomes Used in Combination with Mild Hyperthermia in the Treatment of Local Cancer.</a> Open Nanomed. 3, 38-64 (2011).</li><li>Koning, G. A., Eggermont, A. M., Lindner, L. H., ten Hagen, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperthermia+and+thermosensitive+liposomes+for+improved+delivery+of+chemotherapeutic+drugs+to+solid+tumors.">Hyperthermia and thermosensitive liposomes for improved delivery of chemotherapeutic drugs to solid tumors.</a> Pharm Res. 27 (8), 1750-1754 (2010).</li><li>Viglianti, B. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+monitoring+of+tissue+pharmacokinetics+of+liposome/drug+using+MRI:+illustration+of+targeted+delivery.">In vivo monitoring of tissue pharmacokinetics of liposome/drug using MRI: illustration of targeted delivery.</a> Magn Reson Me. 51 (6), 1153-1162 (2004).</li><li>Ponce, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+imaging+of+temperature-sensitive+liposome+release:+drug+dose+painting+and+antitumor+effects.">Magnetic resonance imaging of temperature-sensitive liposome release: drug dose painting and antitumor effects.</a> J Natl Cancer Ins. 99 (1), 53-63 (2007).</li><li>Kong, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+liposomes+and+hyperthermia+in+a+human+tumor+xenograft+model:+importance+of+triggered+drug+release.">Efficacy of liposomes and hyperthermia in a human tumor xenograft model: importance of triggered drug release.</a> Cancer Res. 60 (24), 6950-6957 (2000).</li><li>Yarmolenko, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+effects+of+thermosensitive+doxorubicin-containing+liposomes+and+hyperthermia+in+human+and+murine+tumours.">Comparative effects of thermosensitive doxorubicin-containing liposomes and hyperthermia in human and murine tumours.</a> Int J Hyperthermia. 26 (5), 485-498 (2010).</li><li>Wang, L. H., Jacques, S. L., Zheng, L. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MCML-Monte+Carlo+modeling+of+photon+transport+in+multi-layered+tissues.">MCML-Monte Carlo modeling of photon transport in multi-layered tissues.</a> Comput Meth Prog Bio. 47, 131-146 (1995).</li><li>Rieke, V., Butts Pauly, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MR+thermometry.">MR thermometry.</a> J Magn Reson Imaging. 27 (2), 376-390 (2008).</li><li>Dou, Y. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat-activated+thermosensitive+liposomal+cisplatin+(HTLC)+results+in+effective+growth+delay+of+cervical+carcinoma+in+mice.">Heat-activated thermosensitive liposomal cisplatin (HTLC) results in effective growth delay of cervical carcinoma in mice.</a> J Control Release. 178, 69-78 (2014).</li><li>Lammers, T., Kiessling, F., Hennink, W. E., Storm, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotheranostics+and+image-guided+drug+delivery:+current+concepts+and+future+directions.">Nanotheranostics and image-guided drug delivery: current concepts and future directions.</a> Mol Pharm. 7 (6), 1899-1912 (2010).</li><li>Lee, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+64Cu-liposomal+PET+agent+(MM-DX-929)+predicts+response+to+liposomal+chemotherapeutics+in+preclinical+breast+cancer+models.">A novel 64Cu-liposomal PET agent (MM-DX-929) predicts response to liposomal chemotherapeutics in preclinical breast cancer models.</a> Thirty-Fifth Annual CTRC-AACR San Antonio Breast Cancer Symposium. , (2012).</li></ol>

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脂质体首先在20世纪60年代作为药物递送载体携带亲水性药物在其内部的水的体积和疏水性药物内的脂质双分子层2开发。除了 ​​在治疗性应用中使用，当标记的放射性核素或装入成像造影剂的脂质体17已探索用于诊断应用。在最近几年，治疗诊断学和治疗诊断对已被进行，以提供用于图像引导的患者分层和药物输送17,18的机会。目前的研究基础上图像引导的药物递送的概...

的实体瘤导致纳米级系统的增强的渗透性和保留（EPR）的病理生理。这导致了许多药物递送系统利用这一效果优点为目标的肿瘤组织，同时最小化的全身副作用1的开发。脂质体递送的技术已被广泛研究用于药物或成像探针2。虽然脂质体已显著降低全身毒性相比常规化疗，已经出现了在临床疗效3,4-一些改进。研究表明，该有限的功效是由于从载体4,5-缺乏药物的释放。其结果是，被激活以释放包封药物响应于外部刺激脂质体的发展已经引起相当大的关注。热疗已经采用了几十年作为一个相对安全的治疗方式对癌症病人6。因此，开发热敏脂质体与热彪作为外部触发已与显著的潜在临床翻译的逻辑组合。事实上，含lysolipid-热敏脂质体多柔比星制剂的，被称为LTSL-DOX，现在已经达到了临床评价7。 与LTSL-Dox最近的临床数据表明，协议热量传输是可以严重影响患者的预后8的关键因素。在人类中，射频，微波，激光和超声换能器被用于局部应用热疗在肿瘤部位9。在临床前研究中，需要皮下肿瘤加热，加热导管10,11和水浴12,13最经常使用的。在该手稿，介绍用于加热使用定制设计的基于激光的加热设置，这使得肿瘤体积的更适形加热皮下肿瘤的新方法。使用MR兼容马terials，设置足够小以适合小动物的MR成像器的孔内，从而允许改变组织温度的实时监测在激光加热过程中。 的MR造影剂，钆特醇（钆-HP-DO3A），被共同封装了CDDP成CDDP（HTLC），称为钆HTLC的热敏脂质体配制剂，用于实时MR图像引导下监测的热和评估活化的药物释放和加热协议的验证。我们的结果表明，基于激光的加热装置中有效地激活包封剂的释放从钆HTLC制剂而经由MR成像监测。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Rotary evaporator</td>
			<td>Heidolph Instruments GmbH &#38; Co.KG</td>
			<td>Laborota 4000</td>
			<td></td>
		</tr>
		<tr>
			<td>High pressure extruder</td>
			<td>Northern Lipids Inc.</td>
			<td>T.001</td>
			<td>10 mL thermobarrel</td>
		</tr>
		<tr>
			<td>Heating circulator</td>
			<td>VWR International LLC.</td>
			<td>11305</td>
			<td>Connected to extruder</td>
		</tr>
		<tr>
			<td>Polycarbonate membrane filter</td>
			<td>Whatman</td>
			<td>110605;110606</td>
			<td></td>
		</tr>
		<tr>
			<td>Differential scanning calorimeter (DSC)</td>
			<td>TA Instruments</td>
			<td>Q100</td>
			<td></td>
		</tr>
		<tr>
			<td>Inductively coupled plasma-atomic emission spectrometer (ICP-AES)</td>
			<td>PerkinElmer</td>
			<td>Optima 7300DV</td>
			<td></td>
		</tr>
		<tr>
			<td>Zetasizer</td>
			<td>Malvern Instruments Ltd.</td>
			<td>Nano-ZS</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell incubator</td>
			<td>NuAire Inc.</td>
			<td>NU-5800</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclip wound clip applier</td>
			<td>Becton Dickinson</td>
			<td>427630</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclip wound clip remover</td>
			<td>Becton Dickinson</td>
			<td>427637</td>
			<td></td>
		</tr>
		<tr>
			<td>Wound clips</td>
			<td>Becton Dickinson</td>
			<td>427631</td>
			<td>9 mm</td>
		</tr>
		<tr>
			<td>763 nm Laser device</td>
			<td>Biolitec</td>
			<td>Ceralas CD 403 laser</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser probe</td>
			<td>Thorlabs Inc.</td>
			<td>FT400EMT</td>
			<td>With SMA and flat cleave connectors</td>
		</tr>
		<tr>
			<td>Spectralon (illuminator)</td>
			<td>Labsphere Inc.</td>
			<td>FAST-SL-5CMX5CM</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>CSTM-SL-5CMX5CM</td>
			<td></td>
		</tr>
		<tr>
			<td>7 Tesla prelinical magnetic resonance (MR) imaging system</td>
			<td>Bruker Corporation</td>
			<td>Biospec 70/30</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber optic temperature sensor</td>
			<td>LumaSense Technologies Inc.</td>
			<td>Luxtron FOT Lab Kit</td>
			<td></td>
		</tr>
		<tr>
			<td>Integrating sphere</td>
			<td>Newport Corporation</td>
			<td>819C</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical power meter</td>
			<td>Newport Corporation</td>
			<td>1830-R</td>
			<td></td>
		</tr>
	</tbody>
</table>

custom-designed laser-based heating apparatus for triggered release of cisplatin from thermosensitive liposomes with magnetic resonance image guidance

脂质体已被用作药物递送系统通过开发增强的渗透性和造成显著减少全身毒性保留（EPR）效果的靶向实体瘤。尽管如此，从脂质体释放包封的药物的不足限制了它们的临床疗效。温度敏感脂质体已被工程化以提供药物的位点特异性释放为了克服有限肿瘤药物生物利用度的问题。我们的实验室已经设计和开发的顺铂的热活化热敏脂质体剂型（顺铂），被称为HTLC，提供顺铂触发释放的实体肿瘤。 体内热活化递送是使用定制的基于激光的加热装置，它提供了保形加热图案在肿瘤部位证实了磁共振温度（MRT）来实现在鼠模型。一种光纤温度监视装置是用来测量在实时的温度在整个采暖期热传递的在线调整交替的激光功率。药物递送被磁共振（MR）图像引导下优化通过共封装的MR造影剂（例如，钆特醇）与顺铂入热敏脂质体作为验证加热协议，并评估肿瘤积累的装置。加热协议包括了5分钟之前HTLC施用和20分钟的加热后喷射的预热期。该加热方案导致有效释放在温水肿瘤观察到相比于未加热的肿瘤和肌肉的最高的MR信号变化的包封剂。这项研究表明基于激光的加热装置，用于临床前热敏脂质体的发展和加热协议用于药物递送的优化的MR引导验证的重要性的成功应用。

的HTLC脂质体使用的是普通的方法，包括脂质膜的形成，水化，挤出和透析制造。在涉及顺铂的步骤，应谨慎采取不暴露CDDP任何铝材，如顺铂将通过一个黑色沉积物的形成被停用。 HTLC的图示示于图3。HTLC所述的物理化学性质总结在最近发表在杂志控释 16的手稿。的钆HTLC制剂的钆和铂浓度为1.87±0.28毫克/ ml和0.10±0.02毫克/毫升。 基...

Watch this Scientific Journal Video about Custom-designed Laser-based Heating Apparatus for Triggered Release of Cisplatin from Thermosensitive Liposomes with Magnetic Resonance Image Guidance at JoVE

Custom-designed Laser-based Heating Apparatus for Triggered Release of Cisplatin from Thermosensitive Liposomes with Magnetic Resonance Image Guidance

的MRI兼容的定制设计的基于激光的加热装置已经开发出来，提供以激活从热敏脂质体释放剂特别在肿瘤区域皮下肿瘤的局部加热。

定制设计的基于激光的加热装置顺铂触发释放的热敏脂质体与磁共振成像制导

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect ...

custom-designed-laser-based-heating-apparatus-for-triggered-release

Research

JoVE Journal

Bioengineering

9.0K 次观看.  University of Toronto. 的MRI兼容的定制设计的基于激光的加热装置已经开发出来，提供以激活从热敏脂质体释放剂特别在肿瘤区域皮下肿瘤的局部加热。 

视频: Custom-designed Laser-based Heating Apparatus for Triggered Release of Cisplatin from Thermosensitive Liposomes with Magnetic Resonance Image Guidance

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

磁共振弹性成像方法评价组织工程构造增长

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

科研

生物工程

Magnetically-Assisted Remote Controlled Microcatheter Tip Deflection under Magnetic Resonance Imaging

X-ray fluoroscopy-guided endovascular procedures have several significant limitations, including difficult catheter navigation and use of ionizing radiation, which can potentially be overcome using a magnetically steerable catheter under MR guidance.
	The main goal of this work is to develop a microcatheter whose tip can be remotely controlled using the magnetic field of the MR scanner. This protocol aims to describe the procedures for applying current to the microcoil-tipped microcatheter to produce consistent and controllable deflections.
	A microcoil was fabricated using laser lathe lithography onto a polyimide-tipped endovascular catheter. In vitro testing was performed in a waterbath and vessel phantom under the guidance of a 1.5-T MR system using steady-state free precession (SSFP) sequencing. Various amounts of current were applied to the coils of the microcatheter to produce measureable tip deflections and navigate in vascular phantoms.
	The development of this device provides a platform for future testing and opportunity to revolutionize the endovascular interventional MRI environment.

Current applied to an endovascular microcatheter with microcoil tip made by laser lathe lithography can achieve controllable deflections under magnetic resonance (MR) guidance, which may improve speed and efficacy of navigation of vasculature during various endovascular procedures.

磁力辅助的远程控制微导管尖端偏转根据磁共振成像

X-ray fluoroscopy-guided  endovascular procedures have several significant limitations, including  difficult catheter navigation and use of ionizing ...

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

交变磁场敏感的混合明胶微凝胶药物控释

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan electron-paramagnetic-resonance (RS-EPR), which can provide quantitative information under in vivo conditions on oxygen concentration, pH, redox status and concentration of signaling molecules (i.e., OH&#8226;, NO&#8226;). The RS-EPR technique has a higher sensitivity, improved spatial resolution (1 mm), and shorter acquisition time in comparison to the standard continuous wave (CW) technique. A variety of phantom configurations have been tested, with spatial resolution varying from 1 to 6 mm, and spectral width of the reporter molecules ranging from 16 &#181;T (160 mG) to 5 mT (50 G). A cross-loop bimodal resonator decouples excitation and detection, reducing the noise, while the rapid scan effect allows more power to be input to the spin system before saturation, increasing the EPR signal. This leads to a substantially higher signal-to-noise ratio than in conventional CW EPR experiments.

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

A new electron paramagnetic resonance (EPR) method, rapid scan EPR (RS-EPR), is demonstrated for 2D spectral spatial imaging which is superior to the traditional continuous wave (CW) technique and opens new venues for in vivo imaging. Results are demonstrated at 250 MHz, but the technique is applicable at any frequency.

快速扫描电子顺磁共振打开新的途径成像生理重要参数<em&gt;在体内</em

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan ...

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

影像引导下，血管源性微流控网络的基于激光的研制

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

Magnetic and Thermal-sensitive Poly(N-isopropylacrylamide)-based Microgels for Magnetically Triggered Controlled Release

Magnetically and thermally sensitive poly(N-isopropylacrylamide) (PNIPAAm)/Fe3O4-NH2 microgels with the encapsulated anti-cancer drug curcumin (Cur) were designed and fabricated for magnetically triggered release. PNIPAAm-based magnetic microgels with a spherical structure were produced via a temperature-induced emulsion followed with physical-crosslinking by mixing PNIPAAm, polyethylenimine (PEI), and Fe3O4-NH2 magnetic nanoparticles. Because of their dispersity, the Fe3O4-NH2 nanoparticles were embedded inside the polymer matrix. The amine groups exposed on the Fe3O4-NH2 and PEI surface supported the spherical structure by physically crosslinking with the amide groups of the PNIPAAm. The hydrophobic anti-cancer drug curcumin can be dispersed in water after encapsulation into the microgels. The microgels were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), and UV-Vis spectral analysis. Furthermore, magnetically triggered release was studied under an external high frequency magnetic field (HFMF). A significant &#34;burst release&#34; of curcumin was observed after applying the HFMF to the microgels due to the magnetic inductive heating (hyperthermia) effect. This manuscript describes the magnetically triggered controlled release of Cur-PNIPAAm/Fe3O4-NH2 encapsulated curcumin, which can be potentially applied for tumor therapy.

Magnetic and Thermal-sensitive PolyN-isopropylacrylamide-based Microgels for Magnetically Triggered Controlled Release

This manuscript describes the preparation of magnetic and thermal-sensitive microgels via a temperature-induced emulsion without chemical reaction. These sensitive microgels were synthesized by mixing poly(N-isopropylacrylamide) (PNIPAAm), polyethylenimine (PEI) and Fe3O4-NH2 nanoparticles for the potential use in magnetically and thermally triggered drug-release.

磁性和热敏性聚（<em&gt;ñ</em基于异丙基丙烯酰胺）的微凝胶用于磁触发控制释放

Magnetically and thermally sensitive poly(N-isopropylacrylamide) (PNIPAAm)/Fe3O4-NH2 microgels with the encapsulated anti-cancer drug curcumin (Cur) were ...

Metabolic Support of Excised, Living Brain Tissues During Magnetic Resonance Microscopy Acquisition

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic resonance (MR) microscopy data. While it is possible to perform MR collections on living, excised mammalian tissue, such experiments have traditionally been constrained by resolution limits and are thus incapable of visualizing tissue microstructure. Conversely, MR protocols that did achieve microscopic image resolution required the use of fixed samples to accommodate the need for static, unchanging conditions over lengthy scan times. The current protocol describes the first available MR technique that enables imaging of living, mammalian tissue samples at microscopic resolutions. Such data is of great importance to the understanding of how pathology-based contrast changes occurring at the microscopic level influence the content of macroscopic MR scans such as those used in the clinic. Once such an understanding is realized, diagnostic methods with greater sensitivity and accuracy can be developed, which will translate directly to earlier disease treatment, more accurate therapy monitoring and improved patient outcomes.
While the described methodology focuses on brain slice preparations, the protocol is adaptable to any excised tissue slice given that changes are made to the gas and perfusate preparations to accommodate the tissue&#39;s specific metabolic needs. Successful execution of the protocol should result in living, acute slice preparations that exhibit MR diffusion signal stability for periods up to 15.5 h. The primary advantages of the current system over other MR compatible perfusion apparatuses are its compatibility with the MR microscopy hardware required to attain higher resolution images and ability to provide constant, uninterrupted flow with carefully regulated perfusate conditions. Reduced sample throughput is a consideration with this design as only one tissue slice may be imaged at a time.

The current protocol describes a method by which users can maintain viability of acute hippocampal and cortical slice preparations during the collection of magnetic resonance microscopy data.

磁共振显微镜下切除活体脑组织的代谢支持

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

含脂肪酸脂质体的制备、纯化及应用

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Apparatus for Harvesting Tissue Microcolumns

This manuscript describes the production process for a laboratory apparatus, made from off-the-shelf components, that can be used to collect microcolumns of full-thickness skin tissue. The small size of the microcolumns allows donor sites to heal quickly without causing donor site scarring, while harvesting full-thickness tissue enables the incorporation of all cellular and extracellular components of skin tissue, including those associated with deeper dermal regions and the adnexal skin structures, which have yet to be successfully reproduced using conventional tissue engineering techniques. The microcolumns can be applied directly into skin wounds to augment healing, or they can be used as the autologous cell/tissue source for other tissue engineering approaches. The harvesting needles are made by modifying standard hypodermic needles, and they can be used alone for harvesting small amounts of tissue or coupled with a simple suction-based collection system (also made from commonly available laboratory supplies) for high-volume harvesting to facilitate studies in large animal models.

Here we describe a protocol for producing harvesting needles that can be used to collect full-thickness skin tissue without causing donor site scarring. The needles can be combined with a simple collection system to achieve high-volume harvesting.

水样组织的采集装置

This manuscript describes the production process for a laboratory apparatus, made from off-the-shelf components, that can be used to collect microcolumns ...

Multi-timescale Microscopy Methods for the Characterization of Fluorescently-labeled Microbubbles for Ultrasound-Triggered Drug Release

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic toxicity and increases circulation time of the drugs. In a novel approach to microbubble-assisted drug delivery, nanoparticles are incorporated in or on microbubble shells, enabling local and triggered release of the nanoparticle payload with ultrasound. A thorough understanding of the release mechanisms within the vast ultrasound parameter space is crucial for efficient and controlled release. This set of presented protocols is applicable to microbubbles with a shell containing a fluorescent label. Here, the focus is on microbubbles loaded with poly(2-ethyl-butyl cyanoacrylate) polymeric nanoparticles, doped with a modified Nile Red dye. The particles are fixed within a denatured casein shell. The microbubbles are produced by vigorous stirring, forming a dispersion of perfluoropropane gas in the liquid phase containing casein and nanoparticles, after which the microbubble shell self-assembles. A variety of microscopy techniques are needed to characterize the nanoparticle-stabilized microbubbles at all relevant timescales of the nanoparticle release process. Fluorescence of the nanoparticles enables confocal imaging of single microbubbles, revealing the particle distribution within the shell. In vitro ultra-high-speed imaging using bright-field microscopy at 10 million frames per second provides insight into the bubble dynamics in response to ultrasound insonation. Finally, nanoparticle release from the bubble shell is best visualized by means of fluorescence microscopy, performed at 500,000 frames per second. To characterize drug delivery in vivo, the triggered release of nanoparticles within the vasculature and their extravasation beyond the endothelial layer is studied using intravital microscopy in tumors implanted in dorsal skinfold window chambers, over a timescale of several minutes. The combination of these complementary characterization techniques provides unique insight into the behavior of microbubbles and their payload release at a range of time and length scales, both in vitro and in vivo.

The presented protocols can be used to characterize the response of fluorescently-labeled microbubbles designed for ultrasound-triggered drug delivery applications, including their activation mechanisms as well as their bioeffects. This paper covers a range of in vitro and in vivo microscopy techniques performed to capture the relevant length and timescales.

用于表征用于超声触发药物释放的荧光标记微气泡的多时间尺度显微镜方法

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic ...