We greatly acknowledge financial support by NIH R01 CA123495, U01 CA151815, R01 CA136841, grants from the Department of Neurosurgery at Cedars-Cedars Medical Center and Arrogene Technology Inc.

所有实验符合官方动物法规，包括在体内进行，且完全按照IACUC协议的手术和非手术方法。 聚苹果酸1。生物生产<ol><li>从琼脂球粒生长的种子培养，并使用25℃的热说明培养箱和基础培养基中8,9疟原虫转移至100毫升培养基中。 </li><li>产生，以避免形成孢子的光下的排除重力包装500毫升微疟原虫的量。 </li><li>准备80克碳酸钙悬浮在8升基本培养基在10升生物反应器容器。传输500毫升装微疟原虫进入安装的反应容器，并允许生物过程为75小时，在25℃，经过滤的空气和由段搅拌器150rpm下搅拌的10升/分钟流量。 </li><li>终止时，将培养液的pH值为4.8，表示生产PMLA的结尾和测量PMLA内容由t他异羟肟酸/铁（III）的测定。 </li><li>冷却液中，以17°C，并允许细胞进行重力沉降。 </li><li>冷却至4℃并调节至pH 7.5，用2M的NaOH中和。通过在该方向上从下到上（粗晶DEAE，用20mM的Tris-HCl pH为7.5，在5℃）填充有1.5升的DEAE-纤维素柱泵上清液。 </li><li>从顶部改变方向至底部后用含有0.3M NaCl的缓冲液3列洗涤并在0.7M的NaCl的存在下洗脱PMLA。 </li><li>调整到0.1M的氯化钙和沉淀PMLA钙80％冰冷乙醇。分馏过葡聚糖凝胶G25到PMLA钙的80 - 300 kDa的，50 - 80 kDa的，和10 - 50 kDa的，使用的水在没有缓冲和盐。 </li><li>通过每个馏分通过一个的Amberlite IR 120H +柱，并立即冷冻流过聚苹果酸在液氮中冷冻干燥。 </li><li>溶解在无水丙酮中。过滤后，除去溶剂在干燥空气流，冷冻干燥和储存在零下20°C。 </li></ol> 2，聚苹果酸为基础的纳米药物的合成<ol><li>通过混合PMLA-H的116毫克（1毫摩尔malyl单位）于1毫升无水丙酮和1毫摩尔N-羟基琥珀酰亚胺和1毫摩尔二环己基在2毫升二甲基甲酰胺（DMF）碳二亚胺活化PMLA（P）的羧基基团。搅拌在20℃3小时。添加0.05的mPEG 5000-NH 2 1毫升DMF其次是0.05毫摩尔三乙胺（TEA）（0.05摩尔％malyl单位）的毫摩尔。 </li><li>加滴加溶解在DMF 0.4毫摩尔亮氨酸乙酯（LOEt）（40摩尔％malyl单位），0.1毫摩尔2 - 巯基-1 - 乙胺（2-MEA）（10摩尔％malyl单位）和0.5毫摩尔的TEA，所有溶解于DMF。每次加入后，允许1小时，检查是否有反应完成由负茚三酮反应（薄层色谱法，薄层色谱法）。 </li><li>不通过用磷酸盐缓冲盐水（30分钟，pH为6.8）自发水解除去剩余的NHS-酯。淡化了帕金森病-10栏，取得“preconjugate”，其为白色粉末通过冷冻干燥。干燥储存在零下20°C。 </li><li>在4.5ml的100mM磷酸钠，150mM的氯化钠，pH值5.5溶解30毫克抗体（mAb，的IgG2a-κ）。 </li><li>减少二硫键用5mM磷酸三（2 - 羧基乙基）膦盐酸盐30分钟，在20℃和净化过PD-10柱。 </li><li>溶解于2ml同样的缓冲液的MAL-PEG 3400-Mal和添加在10毫升最终体积减少的单克隆抗体，并搅拌过夜，在4℃下集中到2.5 mL超过20的Vivaspin，排除30 kD的，净化过葡聚糖凝胶G75。验证过的产品SEC-HPLC和光度测量在280 nm波长处量。 </li><li>附加MAL-PEG-MAL-单抗对“preconjugate”硫醇获得的P / PEG 5000（5％）/ LOEt（40％）/单抗（0.2％）/ 2-MEA（10％）。为此，将5毫升200纳摩尔单克隆抗体-PEG 3400-Mal的50毫克（P / MPEG 5000 / LOEt/2-MEA）在上述缓冲液pH 5.5，调整sulfhy的浓度DRYL〜2毫米和孵化在20℃3小时（收率98％）。 </li><li>溶解3&#39;-H 2 N-AON在DMF / PBS（pH 7.2）中，并与N-琥珀酰亚胺基-3 - （2 -吡啶基二硫）丙酸酯（SPDP）在（1:1 V / V）的DMF /甲醇（茚三酮试验）和纯化的AON-PDP过的Sephadex-LH20在甲醇中，然后在水中和冷冻干燥。 </li><li>孵育800纳摩尔激活的AON在5.5的pH缓冲液与上面制备的MAL-PEG 3400-MAL-单抗preconjugate过夜直至二硫化物交换反应被终止。 </li><li>结合荧光的Alexa Fluor 680 - 马来酰亚胺形成硫醚与聚合物-2-MEA-SH。阻断多余的巯基与3 - 吡啶基丙酸酯（PDP）及净化过葡聚糖凝胶G75。储存于4℃或快速冷冻于-80℃直至进一步使用。 </li></ol> 3，测定和属性<ol><li>通过SEC-HPLC分析进行测试的纯度和稳定性在PBS和血清，在4℃和37℃。使用聚苯乙烯磺酸盐作为molecular体重标准。测量纳米尺寸分布和Zeta-电位。使用商业系统。 </li><li>添加到320微升样品160微升10％（重量/体积）盐酸羟胺和160微升10％（重量/体积）氢氧化钠。经过10-15分钟，拌入160微升5％（W / V）的Fe（Ⅲ）氯3在12％（V / V）HCl和读取氧肟酸/铁的540（III）。计算PMLA假设1毫克/毫升PMLA对应于2.5 A 540。 </li><li>过夜水解纳米药物中的2M盐酸，在110℃和测定苹果酸在反相色谱中，参照样品掺入了苹果酸的标准。裂解纳米药物与100毫米二硫苏糖醇，测量吗啉-AON通过对吗啉AON标准定量反相高效液相色谱法。 </li><li>取纳米药物不裂解，并用蛋白质测定试剂盒测定抗体。量化的mPEG允许其与铵ferrothiocyanate反应，然后提取与氯仿中，在510 nm波长处10读吸光度</SUP&gt;。 </li><li>以每孔检测抗体活性和绑扎5微升的纳米药物（1-10微克单抗）的进行ELISA。用0.5微克/孔的转铁蛋白受体和HER2，分别为板涂覆抗原和过氧化物酶偶联的二抗。 </li><li>对于AON，MPEG和mAb对于苹果酸（100％）计算实现％，与％旨在通过设计比较（参见实施例的表1和表2）。 </li></ol> （4） 在体外测试<ol><li>孵育纳米药物与培养的癌细胞和通过测量它们的活性存活的细胞随着时间的推移，以减少黄试剂[3 - （4,5 - 二甲基吡啶-2 - 基）-5 - （3 - 羧基甲氧基苯基）-2 - （4 - 磺苯基）-2H-四唑，内盐]（MTS），以蓝色染料和读取相对吸光度用光度计。通过该套件的供应商遵循指示。 </li><li>制备细胞提取物在体外或离体进行Western印迹。用冰冷的提取buffer包含SDS和蛋白酶抑制剂的鸡尾酒。分离的蛋白质通过还原性SDS-聚丙烯酰胺凝胶电泳。 </li><li>做免疫印迹为了在样品提取物感兴趣的蛋白质。使用缀合至碱性磷酸酶的第二抗体。 </li><li>使用共聚焦显微镜使用荧光标记以证明细胞摄取和共定位的纳米药物聚合物平台，AON和内涵体。附加的Alexa Fluor 680的纳米复合平台和丽丝胺到的AON作为荧光标签。使用显微镜5X光谱仪和活肿瘤细胞可视化的标记的分子的分布</li><li>与苯乙烯红色荧光染料染色核内体膜和展示内涵体共定位或释放。 </li></ol> 5， 体内试验<ol><li>准备人HER2阳性乳腺癌小鼠模型（裸鼠TAC：铬：（MCR） - Foxnnm）。插入0.72毫克17β-雌二醇释放颗粒（90日发布）■ubcutaneously在每只小鼠的颈部。 </li><li>然后注入1×10 7的BT-474肿瘤细胞皮下注射到右侧，并让肿瘤生长。 </li><li>开始治疗时肿瘤为120 mm 3大小通过注射到尾静脉含有2.5毫克/千克的AON每种抗体和/或4.5毫克/千克150微升纳米缀合物。用于治疗注射，每周两次，共6次。 </li><li>测量肿瘤大小与卡钳两次弱者和使用公式（长×深×宽）×（π/ 6）计算量。后最终注入和镇静和氯胺酮美托咪啶溶液颈椎脱位2周后实施安乐死的动物。 </li><li>分离肿瘤和染色切片用H＆E的形态学评估。 </li><li>为了评估在动物大规模药品流通，使用荧光成像系统。注入的Alexa Fluor 680标记的纳米药物和图像在24和48小时。 </li><li>安乐死的动物和检测氟escence隔离和灌注肿瘤和器官。 </li></ol>

<ol>
	<li>Koboldt, D. C., Fulton, R. S., McLellan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+molecular+portraits+of+human+breast+tumors.+Cancer+Genome+Atlas+Network.">Comprehensive molecular portraits of human breast tumors. Cancer Genome Atlas Network.</a> Nature. 490, 61-70 (2012).</li><li>Kwong, L. N., Costello, J. 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=Oncogenic+NRAS+signaling+differentially+regulates+survival+and+proliferation+in+melanoma.">Oncogenic NRAS signaling differentially regulates survival and proliferation in melanoma.</a> Nat. Med. 18, 1503-1510 (2012).</li><li>Gerlinger, M., Rowan, A. J., Gerlinger, M., Rowan, A. J., 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=Intratumor+heterogeneity+and+branched+evolution+revealed+by+multiregion+sequencing.">Intratumor heterogeneity and branched evolution revealed by multiregion sequencing.</a> N. Engl. J. Med. 366, 883-892 (2012).</li><li>Yap, T. A., Workman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploiting+the+cancer+genome:+strategies+for+the+discovery+and+clinical+development+of+targeted+molecular+therapies.">Exploiting the cancer genome: strategies for the discovery and clinical development of targeted molecular therapies.</a> Annu. Rev. Pharmacol. Toxicol. 52, 549-573 (2012).</li><li>Chatterjee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+Biomarkers:+Knowing+the+Present+and+Predicting+the+Future.">Cancer Biomarkers: Knowing the Present and Predicting the Future.</a> Future Oncol. 1, 37-50 (2005).</li><li>Suh, K. S., Sarojini, S., Youssif, 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=Tissue+Banking,+Bioinformatics,+and+Electronic+Medical+Records:+The+Front-End+Requirements+for+Personalized+Medicine.">Tissue Banking, Bioinformatics, and Electronic Medical Records: The Front-End Requirements for Personalized Medicine.</a> J. Oncology. , (2013).</li><li>Ljubimova, J. Y., Holler, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocompatible+nanopolymers:+the+next+generation+of+breast+cancer+treatment.">Biocompatible nanopolymers: the next generation of breast cancer treatment.</a> Future Medicine, Nanomedicine. 7, 1-4 (2012).</li><li>Lee, B. -. S., Vert, M., Holler, E., Steinb&#252;chel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Water-soluble aiphatic polyesters: poly(malic acid)s. Biopolymers 3a. , 75-103 (2002).</li><li>Lee, B. -. S., Fujita, 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=Polycefin,+a+new+prototype+of+multifunctional+nanoconjugate+based+on+poly(beta-L-malic+acid)+for+drug+delivery.">Polycefin, a new prototype of multifunctional nanoconjugate based on poly(beta-L-malic acid) for drug delivery.</a> Bioconjugate Chem. 17, 317-326 (2006).</li><li>Nag, A., Mitra, 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=A+colorimetric+assay+for+estimation+of+polyethylene+glycol+and+polyethylene+glycolated+protein+using+ammonium+ferrothiocyanate.">A colorimetric assay for estimation of polyethylene glycol and polyethylene glycolated protein using ammonium ferrothiocyanate.</a> Analytical Biochem. 237, 224-231 (1996).</li><li>Ding, H., Inoue, 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=Inhibition+of+brain+tumor+growth+by+intravenous+poly(beta-L-malic)+acid+nanobioconjugate+with+pH-dependent+drug+release.">Inhibition of brain tumor growth by intravenous poly(beta-L-malic) acid nanobioconjugate with pH-dependent drug release.</a> Proc. Natl. Acad. Sci. USA. 107, 18143-18148 (2010).</li><li>Inoue, S., Ding, 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=Nanobioconjugate+inhibition+of+HER2/neu+signaling+and+synthesis+provides+efficient+mouse+breast+cancer+treatment.">Nanobioconjugate inhibition of HER2/neu signaling and synthesis provides efficient mouse breast cancer treatment.</a> Cancer Research. 71, 1454-1464 (2011).</li><li>Maeda, H., Sawa, T., Konno, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+tumor-targeted+delivery+of+macromolecular+drugs,+including+the+EPR+effect+in+solid+tumor+and+clinical+overview+of+the+prototype+polymeric+drug+SMANS.">Mechanism of tumor-targeted delivery of macromolecular drugs, including the EPR effect in solid tumor and clinical overview of the prototype polymeric drug SMANS.</a> J. Control. Release. 74, 47-61 (2001).</li><li>Ljubimova, J. Y., Fujita, M., Ljubimov, Y. J., 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=Poly(malic+acid)+nanoconjugates+containing+various+antibodies+and+oligonucleotides+for+multitargeting+drug+delivery.">Poly(malic acid) nanoconjugates containing various antibodies and oligonucleotides for multitargeting drug delivery.</a> Nanomed. 3, 247-265 (2008).</li><li>Inoue, S., Patil, R., Portilla-Arias, J., 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=Novel+nanobioconjugate+inhibiting+EGFR+expression+in+triple+negative+breast+cancer.">Novel nanobioconjugate inhibiting EGFR expression in triple negative breast cancer.</a> PLoS One. 7, 1-9 (2012).</li><li>Patil, R., Portilla-Arias, J., Ding, 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=Cellular+Delivery+of+Doxorubicin+via+pH-Controlled+Hydrazone+Linkage+Using+Multifunctional+Nano+Vehicle+Based+on+Poly(&#946;-L-malic+Acid).">Cellular Delivery of Doxorubicin via pH-Controlled Hydrazone Linkage Using Multifunctional Nano Vehicle Based on Poly(&#946;-L-malic Acid).</a> Int. J. Mol. Sci. 13, 11681-11693 (2012).</li><li>Patil, R., Portilla-Arias, J., Ding, 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=Temozolomide+delivery+to+tumor+cells+by+a+multifunctional+nano+vehicle+based+on+poly(&#946;-L-malic+acid).">Temozolomide delivery to tumor cells by a multifunctional nano vehicle based on poly(&#946;-L-malic acid).</a> Pharm. Res. 27, 2317-2329 (2010).</li><li>Ljubimova, J. Y., Portilla-Arias, J., Patil, 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=Toxicity+and+efficacy+evaluation+of+multiple+targeted+polymalic+acid+conjugates+for+triple-negative+breast+cancer+treatment.">Toxicity and efficacy evaluation of multiple targeted polymalic acid conjugates for triple-negative breast cancer treatment.</a> J. Drug Target. 21, 956-967 (2013).</li></ol>

作者朱莉娅华Ljubimova，基思·L·布莱克，以及Eggehard奥莱是Arrogene科技股份有限公司股东

实验途径的纳米药物从可生物降解的天然聚合物的制备提出了可在个性化药物的合成中使用。的描述开始，聚苹果酸，这是纳米药物合成一个多功能平台的受控生产和纯化。使用可重复的方法，获得具有高的分子量和在适合于药物合成极端纯度的聚合物。该合成被描述为示出能够有效地治疗HER2阳性乳腺癌的纳米药物。的描述可以被翻译成其他大多数纳米药物治疗癌症的合成。靶向涉及抗体，如赫赛汀结合肿瘤特异性抗原诸如HER2蛋白或任何其他肿瘤标记物被有效地内在化。纳米药物提供了一个选择反义寡核苷酸和化学治疗剂的，这将有效地特雷下抑制癌细胞的生长atment。在这个例子中，赫赛汀结合HER2和具体的反义寡核苷酸退火与HER-2编码的mRNA，导致在HER2信号和重度减低HER2阳性乳腺癌的持续封锁。基于肿瘤靶向和反义寡核苷酸抑制基因表达的基本原则，我们最新合成的其他几个纳米药物，并成功抑制潜伏期人类胶质母细胞瘤和三阴性乳腺癌11,14-18。 合成工作开始于高度纯化的纳米药物的平台，这是从多头绒泡菌 （一个物种的“粘菌”家庭）的培养上清聚苹果酸的制备。制备强调了聚合物的高分子量的，原则上，允许附着大量的抗体，肽，寡核苷酸和其他分子的活性药物输送和肿瘤生长的抑制中起作用。 Followi纳克受控的培养和纯化，聚合物在可预见的产率可再现的质量已经产生。该聚合物被存储方便的条件下进行的任何时间。 的PMLA纳米缀合物与聚合物侧基羧酸的化学活化开始的合成是在几个合成步骤。在步骤之间，合成可任选地被搁置，允许制备的任意数量的中间产物，因此可用于在向上缩放。合成的进展之后是TLC和SEC-HPLC，和纳米药物的两种组合物和活性是由特定组的定量化学分析，酶联免疫吸附，以及各种物理测量的控制。我们的经验是，这些合成已经进展顺利，可重复具有优良的产率和纯度。通过选择抗体和反义寡核苷酸的特异性纳米药物的任何变体可以根据需要在PE是有利合成rsonalized药。 成功治疗人类的HER2阳性乳腺癌的模式是用于制备小鼠癌症模型，应用纳米药物，成像和肿瘤生长的分析有效的代表。 在体外生存力试验的结果是在选择的细胞系和领先的药物在动物实验中使用是有用的。 体内精诺成像验证该纳米药物确实递送到肿瘤。蛋白质印迹分析的结果揭示了是否在预测方式在癌症治疗期间的某些蛋白质的水平响应。肿瘤大小的测量通知关于治疗的成功， 即约抑制作用，衰退或消退。所描述的实施例反映了我们和其它聚苹果酸为基础的纳米药物指示的可预测性，再现性和不存在显着的毒性程度高的经验。对毒性和多个T近期疗效结果argeted聚苹果酸结合物为三阴性乳腺癌的治疗都是在这个概念18的大力支持。一个主要特点是，我们的纳米药物能够穿透生物障碍：内皮屏障由外渗进入肿瘤间质，由核内体摄取的肿瘤细胞膜，并通过亮氨酸乙基酯或trileucine组的动作核内体膜破裂。外渗和核内体摄取被可靠地连接到所述纳米药物的特异性抗体来实现。抗小鼠转铁蛋白受体的抗体介导高效涌入肿瘤由转作用，可能是因为该受体的过度表达于大多数肿瘤脉管系统。不同的抗体附着在纳米相同共轭分子的功能在具体指导纳米药物进入收件人的肿瘤细胞。两种抗体的存在下，在一个用于转作用，另一种为核内体摄取，是最佳的运转是必不可少的。 m的定量分析ALIC酸和抗体强烈建议，以便控制所述纳米药物的最佳组合物。最后，应当指出的是，所有功能于一身的共价纳米药物通过主机脉管到受体的肿瘤细胞提供了药物在化学连接形式的途中 。化学附着使得大多数药物无效的（前体药物），直到它们被从纳米复合平台在目标部位裂解重组为游离药物。这是重要的，因为活化形式提供传送和最小的机会，以唤起有害的副作用时的安全高度。多功能性，有效性和安全性都不错的个性化医学的不可缺少的属性。

在后基因组时代，癌症基因组已被揭开（ 美国国家生物技术研究中心和癌症基因组图谱 ），癌症治疗的未来将占到肿瘤的遗传多样性往往在同一肿瘤1-4。生物信息学和快速的，不买贵的DNA测序允许收购的恶性基因/基因突变在个人层面上2,4,5。一旦该基因已被确定，病人会被个性化医学修改或沉默的恶性6基因表达的治疗。瞄准癌细胞，并提供药物进入这些细胞需要呼吁多功能输送系统。显然，纳米药物可以达到这个要求7。 在发现的怒涛纳米粒子已被证明适合于携带的化疗药物，蛋白质和/或基因的活性物质对癌细胞的有效载荷。然而，不利的影响仍然是广告穿着。其中之一有关的情况下的生物降解性，可能会造成材料的沉积在健康组织和器官惹病的可能性。为了最大限度地减少沉积，我们介绍了无毒和无免疫原性聚苹果酸，这是微生物来源的和可生物降解的，以H 2 O和CO 2 8。我们使用的聚合物，合成的纳米药物的所有功能于一身的共价键类型。它包含化学连接的化疗剂如替莫唑胺，阿霉素，或反义寡核苷酸和服务外渗，组织靶向，溶内体递送的官能团。该药物从本质纳米平台切割时，他们来到了有针对性的肿瘤细胞，从而再生其全部药品的活动。 我们描述了微生物生产的聚合物纳米平台的方法，其纯化，化学合成的纳米药物，它包含曲妥珠单抗（赫赛汀），用于癌症靶向和反义寡核苷酸用于治疗HER2过量的抑制。在应用纳米药物异种人Her2阳性乳腺癌裸鼠，我们证明了癌症治疗的疗效高。肿瘤靶向和基因沉默引入这里供聚苹果酸纳米药物的原理可以在癌症的其他情况下，处理被应用。

<table border="0" cellpadding="0" cellspacing="0" width="935">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Name of Reagent/Material</td>
			<td style="width:263px;">Company</td>
			<td style="width:187px;">Catalog Number</td>
			<td style="width:79px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Mercapto-1-ethylamine (Cysteamine (2MEA)</td>
			<td style="width:263px;">Sigma-Aldrich</td>
			<td style="width:187px;">30078-25G</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#8217;,6-Diamidino-2-phenylindole (DAPI)</td>
			<td colspan="2">Vector Laboratories, Burlingame, CA</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">90-Day release 17&#946;-estradiol pellet&#160;</td>
			<td>Innovative Research of America</td>
			<td style="width:187px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 680 C2 maleimide</td>
			<td style="width:263px;">Invitrogen</td>
			<td style="width:187px;">A20344</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amberlite IR 120H</td>
			<td style="width:263px;">Sigma-Aldrich</td>
			<td style="width:187px;">6428</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Antifoam Y-30 Emulsion</td>
			<td style="width:263px;">Sigam-Aldrich</td>
			<td style="width:187px;">A5758</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Anti-laminin-411 chain mAbs</td>
			<td style="width:263px;">Santa Cruz</td>
			<td style="width:187px;">SC-59980</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Anti-pAkt mAb</td>
			<td style="width:263px;">Cell Signalling</td>
			<td style="width:187px;">9271S</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Anti-PARB mAb</td>
			<td style="width:263px;">BD Biosciences</td>
			<td style="width:187px;">556494</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-von Willebrand Factor antibody&#160;</td>
			<td>Abcam</td>
			<td style="width:187px;">AB6994</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Bacto Yeast Extract</td>
			<td style="width:263px;">Bacto, Dickinson/Sparks, MD</td>
			<td style="width:187px;">212720</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Beta-actin</td>
			<td style="width:263px;">Cell Signalling</td>
			<td style="width:187px;">3700</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BT-474&#160;</td>
			<td style="width:263px;">ATCC</td>
			<td style="width:187px;">HTB-20</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Calcium Carbonate</td>
			<td style="width:263px;">Alfa Aesar</td>
			<td style="width:187px;">36337</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Proliferation Assay kit</td>
			<td>Promega, Madison, WI, USA</td>
			<td>PR-G3580</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centriplus 100&#160;</td>
			<td>Millipore</td>
			<td style="width:187px;">4414</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dicyclohexylcarbodimide</td>
			<td style="width:263px;">Fluka</td>
			<td style="width:187px;">36650</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td style="width:263px;">Sigma-Aldrich</td>
			<td style="width:187px;">D5796</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eosin</td>
			<td style="width:263px;">Cardinal Health</td>
			<td style="width:187px;">S7439-4</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Galardin (MMP-inhibitors)&#160;</td>
			<td style="width:263px;">Santa Cruz</td>
			<td style="width:187px;">SC-994</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GAPDH</td>
			<td style="width:263px;">Cell Signalling&#160;</td>
			<td style="width:187px;">2118</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hematoxilin</td>
			<td style="width:263px;">Cardinal Health</td>
			<td style="width:187px;">S7439-3</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Hemin from porcine</td>
			<td style="width:263px;">Sigma-Aldrich</td>
			<td style="width:187px;">51280</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Herceptin</td>
			<td style="width:263px;">Genentech</td>
			<td style="width:187px;">15534</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Herceptin (Western blotting)</td>
			<td style="width:263px;">Cell Signalling</td>
			<td style="width:187px;">2165S</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">IgG2a-kappa murine malignoma</td>
			<td style="width:263px;">Sigma</td>
			<td style="width:187px;">M77695X5m</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immun-Star AP Substrate Pack</td>
			<td style="width:263px;">Biorad</td>
			<td style="width:187px;">170-5012</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immun-StarTM AP Substrate Pack&#160;</td>
			<td>Biorad</td>
			<td>170-5012</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">LAL Reagent water&#160;</td>
			<td style="width:263px;">Lonza, MD</td>
			<td style="width:187px;">W50-1000</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminin-411 mAbs</td>
			<td style="width:263px;">Abcam</td>
			<td style="width:187px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leucine ethyl ester (LOEt)</td>
			<td style="width:263px;">Sigma-Aldrich</td>
			<td style="width:187px;">61850-10G-F</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Limulus Amebocyte Lysate (LAL) PYROGENT-5000 tests</td>
			<td style="width:263px;">Cambrex BioScience</td>
			<td style="width:187px;">N384</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Lissamin-Morpholino AON</td>
			<td style="width:263px;">Gene Tools</td>
			<td style="width:187px;">Custom made</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-malic acid&#160;</td>
			<td style="width:263px;">Sigma-Aldrich</td>
			<td style="width:187px;">M6413</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Malate dehydrogenase&#160;</td>
			<td style="width:263px;">Sigma-Aldrich</td>
			<td style="width:187px;">M2634</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mal-PEG-Mal</td>
			<td style="width:263px;">Laysan</td>
			<td style="width:187px;">mal-PEG-mal-3400</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel</td>
			<td>BD Biosciences</td>
			<td style="width:187px;">354248</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Milk, nonfat powdered&#160;</td>
			<td style="width:263px;">Proteomics</td>
			<td>M203-10G</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Morpholino oligonucleotides&#160;</td>
			<td style="width:263px;">GeneTools</td>
			<td style="width:187px;">custom made</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">mPEG5000</td>
			<td style="width:263px;">Lysan</td>
			<td style="width:187px;">mPEG-NH2-5000</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-hydroxysuccinimde (NHS)&#160;</td>
			<td style="width:263px;">ACROS Organics</td>
			<td style="width:187px;">15727100</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Ninhydrin</td>
			<td style="width:263px;">Merck</td>
			<td style="width:187px;">1.06762.0100</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitrocellulose Membrane Filter Paper Sandwich</td>
			<td>Invitrogen</td>
			<td>LC2001</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitrocellulose Membrane Filter Paper Sandwich, 0.45 &#181;m&#160;</td>
			<td style="width:263px;">Invitrogen</td>
			<td style="width:187px;">LC2001</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Novex &#174; Tris-Glycine Transfer Buffer (25X)&#160;</td>
			<td>Invitrogen</td>
			<td>LC3675</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Nude Mice [Tac:Cr:(MCr)-Foxnnm]&#160;</td>
			<td>Taconic</td>
			<td style="width:187px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">PBS pH 7.2 10x</td>
			<td style="width:263px;">Gibco</td>
			<td style="width:187px;">10010-49</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">PBS pH 7.2 1x</td>
			<td style="width:263px;">Gibco</td>
			<td style="width:187px;">70013</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PD-10 desalting columns</td>
			<td style="width:263px;">GE Healthcare</td>
			<td style="width:187px;">17-0851</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Physarum polycephalum M3CVII</td>
			<td>ATCC</td>
			<td>204388</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pierce BCA protein assay&#160;</td>
			<td style="width:263px;">Thermo Scientific</td>
			<td style="width:187px;">23225</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease inhibitor cocktail</td>
			<td style="width:263px;">Roche</td>
			<td style="width:187px;">1.18362E+11</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protein Detector ELISA Kit</td>
			<td style="width:263px;">KPL</td>
			<td style="width:187px;">54-62-18</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:263px;"></td>
			<td style="width:187px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sephadex G-25 superfine</td>
			<td style="width:263px;">GE Healthcare</td>
			<td style="width:187px;">17-0031</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sephadex G-75&#160;</td>
			<td style="width:263px;">GE Healthcare</td>
			<td style="width:187px;">17-0050</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sephadex-LH20&#160;</td>
			<td style="width:263px;">GE Healthcare</td>
			<td style="width:187px;">17-0090</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SKBR-3&#160;</td>
			<td style="width:263px;">ATCC</td>
			<td style="width:187px;">HTB-30</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SPDP</td>
			<td style="width:263px;">Proteochem&#160;</td>
			<td style="width:187px;">C1116</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streamline-DEAE</td>
			<td style="width:263px;">GE Healthcare</td>
			<td style="width:187px;">17-0994</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Styryl Red FM 1-43&#160;</td>
			<td>Life Technologies&#160;</td>
			<td style="width:187px;">T-3163</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:263px;"></td>
			<td style="width:187px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TBS 10x</td>
			<td>Bio-Rad</td>
			<td>170-6435</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TCEP</td>
			<td style="width:263px;">Sigma-Aldrich</td>
			<td style="width:187px;">C4706-2G</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TLC, silica coated aluminia sheets</td>
			<td style="width:263px;">Merck, Darmstadt, DE</td>
			<td style="width:187px;">60F254</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trileucine (LLL)</td>
			<td style="width:263px;">Bachem</td>
			<td style="width:187px;">H-3915</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-114&#160;</td>
			<td style="width:263px;">Sigma-Aldrich</td>
			<td style="width:187px;">X114</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween&#174;20&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:263px;"></td>
			<td style="width:187px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vivaspin 20&#160;</td>
			<td style="width:263px;">VWR</td>
			<td style="width:187px;">14005-302</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:407px;">DAPI</td>
			<td style="width:263px;">Vector Laboratories, Burlingame, CA</td>
			<td style="width:187px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dexmedetomidine</td>
			<td style="width:263px;">Pfizer</td>
			<td style="width:187px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Atipamezole</td>
			<td style="width:263px;">Pfizer</td>
			<td style="width:187px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carprofen</td>
			<td style="width:263px;">Pfizer</td>
			<td style="width:187px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Betadine</td>
			<td style="width:263px;">Foster and Smith, Wisconsin</td>
			<td style="width:187px;"></td>
			<td style="width:79px;"></td>
		</tr>
	</tbody>
</table>

polymalic acid-based nano biopolymers for targeting of multiple tumor markers: an opportunity for personalized medicine?

Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this diversity, personalized medicine is developed for silencing malignancy associated genes. Nano drugs fit these needs by targeting tumor and delivering antisense oligonucleotides for silencing of genes. As drugs for the treatment are often administered repeatedly, absence of toxicity and negligible immune response are desirable. In the example presented here, a nano medicine is synthesized from the biodegradable, non-toxic and non-immunogenic platform polymalic acid by controlled chemical ligation of antisense oligonucleotides and tumor targeting molecules. The synthesis and treatment is exemplified for human Her2-positive breast cancer using an experimental mouse model. The case can be translated towards synthesis and treatment of other tumors.

人类的HER2阳性乳腺癌通过沉默HER2受体的表达和阻断HER2-12的信号抑制 战略 在不同形式的人类乳腺癌，HER2阳性肿瘤有最差的临床结果。我们目前的成功治疗人类HER-2过表达乳腺癌的裸鼠模型。该策略涉及纳米药物活性的HER2信号通路使用赫赛汀和HER-2特异性AON用于阻断该受体的HER-2的mRNA依赖性合成（ 图1）两者的直接沉默。引线纳米缀合物，其中含有曲妥珠单抗和抗HER2 AON示于图3以及两个控制哪些是不含任何赫赛汀或AON的。先导化合物含有抗MsTfRmAb由转作用来实现主动外渗救援人员到场唉结合内皮转铁蛋白受体的肿瘤血管。摄取到肿瘤小得多有人指出在不存在抗体和归因于肿瘤相关的EPR效应13。合成的影像学检查包含的Alexa Fluor 680纳米偶联物的荧光版本。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/50668/50668fig1highres.jpg" width="500" /> 图1机构AON递送到HER2阳性乳腺癌细胞和抑制肿瘤生长的左上角：改编自图3纳米结合。左下角：表面上表达转铁蛋白受体小鼠肿瘤血管。纳米药物与受体结合并通过胞转进入肿瘤。此外，在某种程度上获得的肿瘤是可能通过参与肿瘤依赖性增强的摄取和retenti无序内皮层上（EPR）13。接下来，纳米药物结合HER2的表达对人癌细胞和内化到早期内涵体。结合还阻止HER2信号通路。内涵体的成熟和其酸化后，纳米药物的核内体逃逸机制被激活。纳米药物进入细胞质从纳米平台公布的二硫化物间隔的还原裂解，并结合HER-2基因阻断HER2的合成。阻挡合成的延伸阻断HER2-信号传导的和诱导的肿瘤生长抑制。 微生物生产的聚苹果酸平台 纳米复合平台，聚苹果酸（PMLA），通过多头绒泡菌的培养microplasmodia和从作为协议中描述的和在图2所示的液体培养基的纯化制备。生产和纯化光滑，重现性好。重要的是要续<html> ROL时间，pH值和温度，以避免聚合物的自发裂解。小心洗涤和DEAE-柱的洗脱建议为了去除DNA，碳水化合物，毒素和有色物质。溶解在无水丙酮中，除去不溶物后，获得极端纯度。生产PMLA的总金额为每个广告系列5±1克1.5±0.5克金额高度纯化的M PMPLA 瓦特 80,000-100,000正在使用的10升生物反应器时，可重复获得。 SEC-HPLC洗脱曲线是对称的。根据数量分布动态光散射分析表明对应于8±1纳米的流体动力学直径的单峰和PDI的多分散性指数）=（0.10±0.02，通过仪器软件，用于假定的球形颗粒计算。 ζ电位为-22±2毫伏（pH值7.5）。 / 50668/50668fig2highres.jpg“宽度=”500“/&gt; 图2。生产和多头绒泡菌的培养微疟原虫聚苹果酸（改编自Lee 等人。9）的净化 。聚苹果酸（PMLA）是从多头绒泡菌的疟原虫产生在一个生物反应器，并通过在阴离子交换剂（DEAE）结合从培养上清液回收。洗脱剂是乙醇沉淀钙盐。钙盐的解决方案是分子量分级分离过的Sephadex-G25。 PMLA含有馏分从钙的盐转化成酸过的Amberlite IR-120H +。免费PMLA最后冷冻干燥，得到干燥的白色粉末。 化学合成的纳米导线的共轭 P /的mPEG 5000（5％）/ LOEt（40％）/ AON（2.5％）/赫赛汀（0.2％）/一<html> NTI-MsTfRmAb（0.2％） 两种前体偶联物的纳米导线的纳米药物及的示意性结构示于图由图3。领头的包含所有成分，而前体被设计成缺乏要么AON或HER2抗体赫赛汀。除了​​AON和单克隆抗体，所述化合物包含聚乙二醇，MPEG 5000，以减少与血浆蛋白结合，通过网状内皮系统（RES），以及降解的酶促切割的间隙。反义序列5&#39;-CAT-GGT-GCT-CAC-TGC-GGC-TCC-GGC-3&#39;是互补的Her2表达，并已在体外被仔细测试了几种表达HER2的细胞系，以获得较高的特异性朝阻断HER2合成，尚未出现脱靶效应。 8/50668fig3highres.jpg“宽度=”500“/&gt; 图3纳米结合物来治疗人类的HER2阳性乳腺癌 。领先的纳米药物（顶结构）包含两种药物（赫赛汀，AON HER2）。版本2和3仅包含一个药物。摄取1和2为人类HER-2过表达的肿瘤细胞是通过曲妥珠单抗的结合来管理。纳米共轭3，其中不含有曲妥珠单抗，由人体细胞接受抗HuTfRmAb为核内体摄取。的Alexa Fluor 680缀合是可选的用于成像目的。红色条代表共轭纳米平台PMLA / LOEt（40％）。 ％表示所消耗的指示的配体（总的在纳米药物= 100％苹果酸残基的量）的结合苹果酸残基的比例。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/50668/50668fig4highres.jpg" width="500" /> 图4。化学合成纳米结合物的P / LOEt / AON HER2 / anti-MsTfR/Herceptin从上到下：。的挂件羧酸盐（化学活化）的PMLA-N-羟基琥珀酰亚胺酯（PMLA-NHS）的合成。更换NHS通过形成酰胺与2 -巯基-1 - aminoethan（2-MEA），甲基-PEG 5000 -胺（MPEG 5000-NH 2），trileucine（LLL）或亮氨酸乙基酯（LOEt）。这种“preconjugate”重视单抗-MAL-PEG-Mal的由硫醚形成和AON通过形成剪切的二硫键。剩余的2-MEA被盖住形成酰氨基乙基 - 二硫代丙酸。只显示附件的单一单抗的。多个附件通过使用单克隆抗体的混合物进行管理。百分比％表示羧酸盐必然不同配体（100％免费PMLA）的分数。 合成了纳米偶联物如在图URE 4中概述。引线的计算分子量共轭是719,000。合成的总收率为45±5％，对于苹果酸含量。上平均的100 kDa的PMLA平台862 malyl单位（= 100％），40％进行LOEt，5％的mPEG，2％的AON 2％，0.2％曲妥珠单抗和0.2％抗MsTfR单抗。量为每个抗体对应于平均每PMLA平台分子1.7分子。在％设计和组分析验证了％是相同的±5％以内。一个例子是在表1中对苹果酸，AON，单克隆抗体和MPEG 5000，并在表2的实验内容的计算示出了由设计的内容进行比较的情况。按照根据第3条件的高纯度，高反应产率和通过尺寸排阻高效分离（ 例如游离mAb和mAb-nanoconjugate是通过在SEC-HPLC 1分钟间隔），和在溶剂中的选择性溶解性的基础上实现的。单克隆抗体的活性为<html>示在整个纳米药物的合成要保留的，并且证实了这两种抗体分别组装在同一聚合物平台上。非典型的酶联免疫吸附试验的结果示于图URE 5。在一般情况下，该测定法进行定量分析组为苹果酸，AON，蛋白质，聚乙二醇和用于ELISA是健壮的，当由不同的人员和仪器不仅在所报告的合成的情况下进行的，得到可重复的结果，但也可用于分析时其他合成nanoconjugates。 <img alt="表1" fo:content-width="6in" src="/files/ftp_upload/50668/50668table1.jpg" width="600" /> 表1。计算参照苹果酸含量纳米药物组合物。 668table2.jpg“宽度=”600“/&gt; 表2的比较与设计构图的实验纳米药物组成的。 <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/50668/50668fig5highres.jpg" width="500" /> ELISA图5。对抗体亲和力的测定化学合成后，对于示范多个结合不同抗体的该药物的纳米抗体包含单克隆抗体（A）和抗体单克隆抗体（B）对抗原A和B，分别。 ELISA板包被抗原（A）。免费单抗（A），游离mAb（B）和纳米药物应用于酶联免疫吸附板。洗涤后，将抗体与过氧化物酶二级特异性针对任一单克隆抗体（A）或单克隆抗体（B）的偶联的抗体进行测试，而只有单克隆抗体（A）被绑定到抗原（A）。可以看出，自由和纳米药物缀合的mAb（A），和​​间接缀合相同的纳米药物分子的单克隆抗体（B），但不游离mAb（B）的，被保留在板上。的浓度依赖性表明可比的结合亲和力为游离和偶联的单克隆抗体（A），表示该化学缀合并不影响抗体的结合活性。此外，在同一个物理实体（Nano平台）的结果在抗体单抗（B）检测共连接单抗（B）与单克隆抗体（A）。这里所示的实验结果指抗人TfRmAb（A）和抗小鼠TfRmAb（B）。类似的结果已经显示出对测试的其他抗体游，诸如抗MsTfR的mAb和抗HER2单克隆抗体（赫赛汀）。 理化调查表明流体动力学直径22.1±2.3 nm的用于P / MPEG（5％）/ LOEt（40％）/ AON（2％）/赫赛汀（0.2％）/抗MsTfRmAb（0.2％）（铅，二药物版），20.1±为P / MPEG（5％）/ LOEt（40％）/ AON（2％）/抗MsTfRmAb（0.2％）/抗HuTfRmAb（0.2％）（AON的药物版）2.4纳米和15.1±1.2 nm的用于P / MPEG（5％）/ LOEt（40％）/赫赛汀（0.2％）（赫赛汀药物的版本）。 Z-电位在pH = 7.5均按此顺序-5.2±0.4 mV时，-5.7±0.6 mV的，和-4.1±0.4 mV的。应当提及，纳米缀合物的测定的流体动力学直径没有按照相加测定游离组分的直径。 通过肿瘤细胞膜输送的纳米药物和释放到细胞质后0小时和3小时 Ř 纳米药物通过细胞膜由核内体摄取递送示于图URE 6 0小时和3小时后。荧光标签附着在纳米药物的平台（的Alexa Fluor 680，绿色）和AON（丽丝胺，红色）。它们的叠加显示在面板D＆L，连同标有内涵体（蓝色）在图G＆O的荧光Pearson相关系数（R（R）的图像关于平台/核内体的共定位，AON /内涵体，平台/ AON为0小时和3小时。t分别计算他3小时的图像显示释放从内涵体到AON的从纳米药物通过在细胞质中谷胱甘肽依赖性二硫化物断开的细胞质和分离。 <img alt="图6" fo:content-width="5in" src="/files/ftp_upload/50668/50668fig6highres.jpg" width="500" /> 图6。共聚焦显微镜对纳米共轭吸收的靶细胞（改编自丁等 11）。细胞孵育30分钟，在37℃下用纳米药物，其已在该聚合物平台，并与丽丝胺（红色）连接到AON被双标记的Alexa Fluor 680（绿色）。此外，内体进行染色的FM1-43（蓝色）。 0小时（A）和3小时（B）的培养后的本地化所示。图A，AB，＆B，IJ染色显示仅由纳米荧光共轭，并他们共定位于A，CD＆B，KL。此外，核内体染色显示在面板A，E＆B，m和纳米共轭和endosomesin板A，的共定位FG＆B，没有 。图A，H＆B，p显示相衬的各个面板（C）Pearson相关系数R（R）为平台的内涵体，AON-内体，平台-AON计算0小时和3小时的共定位。 <a href="https://www.jove.com/files/ftp_upload/50668/50668fig6large.jpg" target="_blank">请点击此处查看这个数字的放大版本。</a> 人类乳腺癌的体外和体内抑制 在 BT-474 HER2过度表达的细胞系中的COM 体外生长的抑制作用型坯与MDA-MB-231低表达细胞系示于图URE 7。抑制度分别为50％和30％，由引线的纳米复合P / MPEG / LOEt / AON HER2 /赫赛汀/抗MsTfRmAb。抑制由其它化合物是以下但高于对BT-474比对MDA-MB-231细胞。 BT-474细胞系被选定为异种小鼠乳腺癌的治疗。 <img alt="图7" fo:content-width="5in" src="/files/ftp_upload/50668/50668fig7highres.jpg" width="500" /> 图7。 在体外生长的HER2过度表达的BT-474乳腺癌细胞和低表达的MDA-MB-231细胞（改编自井上等人 12）的生长抑制的HER2过度表达的BT-474乳腺癌细胞系的比较（ 抑制左）和HER2低表达的乳腺癌细胞系MDA-MB-231（右）。转铁蛋白受体（S）表示反MsTfRmAB和转铁蛋白受体（胡/女士）代表反HuTfRmAb及抗MsTfRmAb。引线纳米药物，P / MPEG / LOEt / AON HER2 /赫赛汀/转铁蛋白受体（MS），和纳米药物缺乏或赫赛汀或AON的HER2用PBS，赫赛汀和AON HER2比较。在不存在曲妥珠单抗，抗MsTfRmAb缀合的肿瘤细胞，以提供核内体摄取。浓度分别为40微克/毫升就抗体，4μM关于AON HER2，4微米endoporter（应用的AON）。意义表示为*，P &lt;0.05 ** P &lt;0.02 *** P &lt;0.003，用PBS进行比较。 在体内治疗中的图8的结果表示为小鼠的BT-474过表达HER2的乳腺癌。增长是由含赫赛汀和HER-2特异性AON相比，只用PBS（URE图8）处理的对照引纳米药物抑制&gt; 95％。抑制为60％或更少与Herceptin单独，P / MPEG / LOEt /赫赛汀或P / MPEG / LOEt / AON /抗MsTfRmAb /抗HuTfRmAb。肿瘤提取物的Western blot分析表明抑制HER2两者合成和Akt的磷酸化，但没有改变总Akt和看家酶GAPDH的。 PARP裂解对应于细胞凋亡的增加的水平。治疗和组织切片染色用H＆E示出肿瘤消退后的图片的肿瘤示于图URE 9。 <img alt="图8" fo:content-width="5in" src="/files/ftp_upload/50668/50668fig8highres.jpg" width="500" /> 与铅和前体的纳米药物（改编自Inoue 等 12）在治疗过程中图8。肿瘤大小和蛋白质。一 ，肿瘤大小为各种治疗（21天至第42天，共8次注射）。线表示标准偏差的平均值。结合具有抗Her2的 AON和赫赛汀的铅优于要么赫赛汀单独或单含纳米药物结合物：B。对HER2，磷酸化Akt，总Akt，PARB表达的各种治疗方法的效果，切割PARB和GAPDH显示免疫印迹。 <a href="https://www.jove.com/files/ftp_upload/50668/50668fig8large.jpg" target="_blank">请点击这里查看这个数字的放大版本。</a> <img alt="图9" fo:content-width="5in" src="/files/ftp_upload/50668/50668fig9highres.jpg" width="500" /> HER2阳性BT-474人乳腺癌裸鼠的图9。治疗。肿瘤消退和坏死/凋亡的组织切片（改编自Ionue 等人 12）。鞋面：肿瘤（红色箭头）的收缩。指示雌激素的颗粒，以支持肿瘤生长（蓝色箭头）。罗威记：肿瘤切片苏木精和曙红（H＆E）染色。增殖的肿瘤表现出的紧密堆积的肿瘤细胞。坏死组织被认为是其中的肿瘤细胞已被淘汰。 <a href="https://www.jove.com/files/ftp_upload/50668/50668fig9large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine? at JoVE.com

Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine?

基于聚苹果酸的纳米药物的一个例子，提出对个性化的药物，适用于癌症的合理设计。它描述了一种纳米药物来治疗HER2阳性乳腺癌裸鼠的合成。

聚苹果酸为基础的生物聚合物纳米靶向的多种肿瘤标志物：用于个性化医疗的机会？

Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this ...

polymalic-acid-based-nano-biopolymers-for-targeting-multiple-tumor

Research

JoVE Journal

Chemistry

16.5K 次观看.  Cedars-Sinai Medical Center. 基于聚苹果酸的纳米药物的一个例子，提出对个性化的药物，适用于癌症的合理设计。它描述了一种纳米药物来治疗HER2阳性乳腺癌裸鼠的合成。 

视频: Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine?

Glycan Node Analysis: A Bottom-up Approach to Glycomics

Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and extracellular events. Many pathological conditions, notably cancer, affect gene expression, which can in turn deregulate the relative abundance and activity levels of glycoside hydrolase and glycosyltransferase enzymes. Unique aberrant whole glycans resulting from deregulated glycosyltransferase(s) are often present in trace quantities within complex biofluids, making their detection difficult and sometimes stochastic. However, with proper sample preparation, one of the oldest forms of mass spectrometry (gas chromatography-mass spectrometry, GC-MS) can routinely detect the collection of branch-point and linkage-specific monosaccharides ("glycan nodes") present in complex biofluids. Complementary to traditional top-down glycomics techniques, the approach discussed herein involves the collection and condensation of each constituent glycan node in a sample into a single independent analytical signal, which provides detailed structural and quantitative information about changes to the glycome as a whole and reveals potentially deregulated glycosyltransferases. Improvements to the permethylation and subsequent liquid/liquid extraction stages provided herein enhance reproducibility and overall yield by facilitating minimal exposure of permethylated glycans to alkaline aqueous conditions. Modifications to the acetylation stage further increase the extent of reaction and overall yield. Despite their reproducibility, the overall yields of N-acetylhexosamine (HexNAc) partially permethylated alditol acetates (PMAAs) are shown to be inherently lower than their expected theoretical value relative to hexose PMAAs. Calculating the ratio of the area under the extracted ion chromatogram (XIC) for each individual hexose PMAA (or HexNAc PMAA) to the sum of such XIC areas for all hexoses (or HexNAcs) provides a new normalization method that facilitates relative quantification of individual glycan nodes in a sample. Although presently constrained in terms of its absolute limits of detection, this method expedites the analysis of clinical biofluids and shows considerable promise as a complementary approach to traditional top-down glycomics.

This article presents an enhanced form of a novel bottom-up glycomics technique designed to analyze the pooled compositional profile of glycans in unfractionated biofluids through the chemical breakdown of glycans into their constituent linkage-specific monosaccharides for detection by GC-MS. Potential applications include early detection of cancer and other glycan-affective disorders.

聚糖节点分析：自下而上的方法，以糖原组学

Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and ...

科研

化学

Highly Stable, Functional Hairy Nanoparticles and Biopolymers from Wood Fibers: Towards Sustainable Nanotechnology

Nanoparticles, as one of the key materials in nanotechnology and nanomedicine, have gained significant importance during the past decade. While metal-based nanoparticles are associated with synthetic and environmental hassles, cellulose introduces a green, sustainable alternative for nanoparticle synthesis. Here, we present the chemical synthesis and separation procedures to produce new classes of hairy nanoparticles (bearing both amorphous and crystalline regions) and biopolymers based on wood fibers. Through periodate oxidation of soft wood pulp, the glucose ring of cellulose is opened at the C2-C3 bond to form 2,3-dialdehyde groups. Further heating of the partially oxidized fibers (e.g., T = 80 &#176;C) results in three products, namely fibrous oxidized cellulose, sterically stabilized nanocrystalline cellulose (SNCC), and dissolved dialdehyde modified cellulose (DAMC), which are well separated by intermittent centrifugation and co-solvent addition. The partially oxidized fibers (without heating) were used as a highly reactive intermediate to react with chlorite for converting almost all aldehyde to carboxyl groups. Co-solvent precipitation and centrifugation resulted in electrosterically stabilized nanocrystalline cellulose (ENCC) and dicarboxylated cellulose (DCC). The aldehyde content of SNCC and consequently surface charge of ENCC (carboxyl content) were precisely controlled by controlling the periodate oxidation reaction time, resulting in highly stable nanoparticles bearing more than 7 mmol functional groups per gram of nanoparticles (e.g., as compared to conventional NCC bearing &#60;&#60; 1 mmol functional group/g). Atomic force microscopy (AFM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) attested to the rod-like morphology. Conductometric titration, Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), dynamic light scattering (DLS), electrokinetic-sonic-amplitude (ESA) and acoustic attenuation spectroscopy shed light on the superior properties of these nanomaterials.

Synthesis schemes to prepare highly stable wood fiber-based hairy nanoparticles and functional cellulose-based biopolymers have been detailed.

高稳定性，功能性毛纳米粒子和木纤维生物聚合物：迈向可持续纳米技术

Nanoparticles, as one of the key materials in nanotechnology and nanomedicine, have gained significant importance during the past decade. While ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

蛋白质生物缀合物的合成<em&gt;通过</em&gt;半胱氨酸马来酰亚胺化学

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Low-energy Cathodoluminescence for (Oxy)Nitride Phosphors

Nitride and oxynitride (Sialon) phosphors are good candidates for the ultraviolet and visible emission applications. High performance, good stability and flexibility of their emission properties can be achieved by controlling their composition and dopants. However, a lot of work is still required to improve their properties and to reduce the production cost. A possible approach is to correlate the luminescence properties of the Sialon particles with their local structural and chemical environment in order to optimize their growth parameters and find novel phosphors. For such a purpose, the low-voltage cathodoluminescence (CL) microscopy is a powerful technique. The use of electron as an excitation source allows detecting most of the luminescence centers, revealing their luminescence distribution spatially and in depth, directly comparing CL results with the other electron-based techniques, and investigating the stability of their luminescence properties under stress. Such advantages for phosphors characterization will be highlighted through examples of investigation on several Sialon phosphors by low-energy CL.

Low-energy Cathodoluminescence for OxyNitride Phosphors

An excellent chemical and luminescence stabilities of (oxy)nitride phosphors present it as an promising alternative to currently used sulfide and oxide phosphors. In this paper, we present the way to investigate its local luminescence properties using low-energy cathodoluminescence (CL).

低能量的阴极射线（氧）氮化物荧光粉

Nitride and oxynitride (Sialon) phosphors are good candidates for the ultraviolet and visible emission applications. High performance, good stability and ...

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the inverse electron demand Diels-Alder (IEDDA) cycloaddition between a radiolabeled tetrazines and trans-cyclooctene (TCO) linked to a biomolecule has proven to be a highly effective bioorthogonal approach to imaging specific biological targets. Despite the fact that technetium-99m remains the most widely used isotope in diagnostic nuclear medicine, there is a scarcity of methods for preparing 99mTc-labeled tetrazines. Herein we report the preparation of a family of tridentate-chelate-tetrazine derivatives and their Tc(I) complexes. These hitherto unknown compounds were radiolabeled with 99mTc using a microwave-assisted method in 31% to 83% radiochemical yield. The products are stable in saline and PBS and react rapidly with TCO derivatives in vitro. Their in vivo pre-targeting abilities were demonstrated using a TCO-bisphosphonate (TCO-BP) derivative that localizes to regions of active bone metabolism or injury. In murine studies, the 99mTc-tetrazines showed high activity concentrations in knees and shoulder joints, which was not observed when experiments were performed in the absence of TCO-BP. The overall uptake in non-target organs and pharmacokinetics varied greatly depending on the nature of the linker and polarity of the chelate.

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Here, we describe a protocol for radiolabeling and in vivo testing of tridentate 99mTc(I) chelate-tetrazine derivatives for pre-targeting and bioorthogonal chemistry.

制备和评价<sup&gt;99米</sup&gt;锝标记的三齿螯合物预靶向使用生物正交化学

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the ...

An Aptamer-based Sensor for Unchelated Gadolinium(III)

A method for determining the presence of unchelated trivalent gadolinium ion (Gd3+) in aqueous solution is demonstrated. Gd3+ is often present in samples of gadolinium-based contrast agents as a result of incomplete reactions between the ligand and the ion, or as a dissociation product. Since the ion is toxic, its detection is of critical importance. Herein, the design and usage of an aptamer-based sensor (Gd-sensor) for Gd3+ are described. The sensor produces a fluorescence change in response to increasing concentrations of the ion, and has a limit of detection in the nanomolar range (~100 nM with a signal-to-noise ratio of 3). The assay may be run in an aqueous buffer at ambient pH (~7 - 7.4) in a 384-well microplate. The sensor is relatively unreactive toward other physiologically relevant metal ions such as sodium, potassium, and calcium ions, although it is not specific for Gd3+ over other trivalent lanthanides such as europium(III) and terbium(III). Nevertheless, the lanthanides are not commonly found in contrast agents or the biological systems, and the sensor may therefore be used to selectively determine unchelated Gd3+ in aqueous conditions.

An Aptamer-based Sensor for Unchelated GadoliniumIII

The use of polydeoxynucleotide (44-mer aptamer) molecules for sensing unchelated gadolinium(III) ion in an aqueous solution is described. The presence of the ion is detected via an increase in the fluorescence emission of the sensor.

对于未螯合钆基于核酸适配体传感器（III）

A method for determining the presence of unchelated trivalent gadolinium ion (Gd3+) in aqueous solution is demonstrated. Gd3+ is often present in samples ...

An Engineered Split-TET2 Enzyme for Chemical-inducible DNA Hydroxymethylation and Epigenetic Remodeling

DNA methylation is a stable and heritable epigenetic modification in the mammalian genome and is involved in regulating gene expression to control cellular functions. The reversal of DNA methylation, or DNA demethylation, is mediated by the ten-eleven translocation (TET) protein family of dioxygenases. Although it has been widely reported that aberrant DNA methylation and demethylation are associated with developmental defects and cancer, how these epigenetic changes directly contribute to the subsequent alteration in gene expression or disease progression remains unclear, largely owing to the lack of reliable tools to accurately add or remove DNA modifications in the genome at defined temporal and spatial resolution. To overcome this hurdle, we designed a split-TET2 enzyme to enable temporal control of 5-methylcytosine (5mC) oxidation and subsequent remodeling of epigenetic states in mammalian cells by simply adding chemicals. Here, we describe methods for introducing a chemical-inducible epigenome remodeling tool (CiDER), based on an engineered split-TET2 enzyme, into mammalian cells and quantifying the chemical inducible production of 5-hydroxymethylcytosine (5hmC) with immunostaining, flow cytometry or a dot-blot assay. This chemical-inducible epigenome remodeling tool will find broad use in interrogating cellular systems without altering the genetic code, as well as in probing the epigenotype&#8722;phenotype relations in various biological systems.

Detailed protocols for introducing an engineered split-TET2 enzyme (CiDER) into mammalian cells for chemical inducible DNA hydroxymethylation and epigenetic remodeling are presented.

用于化学诱导的 DNA 化和表观遗传重塑的基因工程 Split-TET2 酶

DNA methylation is a stable and heritable epigenetic modification in the mammalian genome and is involved in regulating gene expression to control ...

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent proteins and generation of fluorescent fusion proteins have been wildly used as an effective tool to directly visualize the protein localization and dynamics in cells. It is especially useful to compare them with well-known organelle markers after co-expression with the protein of interest. Nevertheless, classical approaches for protein co-expression in plants usually involve multiple independent expression plasmids, and therefore have drawbacks that include low co-expression efficiency, expression-level variation, and high time expenditure in genetic crossing and screening. In this study, we describe a robust and novel method for co-expression of multiple chimeric fluorescent proteins in plants. It overcomes the limitations of the conventional methods by using a single expression vector that is composed of multiple semi-independent expressing cassettes. Each protein expression cassette contains its own functional protein expression elements, and therefore it can be flexibly adjusted to meet diverse expression demand. Also, it is easy and convenient to perform the assembly and manipulation of DNA fragments in the expression plasmid by using an optimized one-step reaction without additional digestion and ligation steps. Furthermore, it is fully compatible with current fluorescent protein derived bio-imaging technologies and applications, such as FRET and BiFC. As a validation of the method, we employed this new system to co-express fluorescently fused vacuolar sorting receptor and secretory carrier membrane proteins. The results show that their perspective subcellular localizations are the same as in previous studies by both transient expression and genetic transformation in plants.

We have developed a novel method for co-expressing multiple chimeric fluorescent fusion proteins in plants to overcome the difficulties of conventional methods. It takes advantage of using a single expression plasmid that contains multiple functionally independent protein expressing cassettes to achieve protein co-expression.

一种高效的植物多嵌合体荧光融合蛋白的协同表达

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent ...

Iron Nanowire Fabrication by Nano-Porous Anodized Aluminum and its Characterization

Magnetic nanowires possess unique properties that have attracted the interest of different fields of research, including basic physics, biomedicine, and data storage. We demonstrate a fabrication method for iron (Fe) nanowires&#160;via electrochemical deposition into anodic&#160;alumina oxide (AAO) templates. The templates are fabricated by anodization of aluminum (Al) discs, and the pore length and diameter are controlled by changing the anodizing conditions. Pores with an average diameter of around 120 nm are created using oxalic acid as the electrolyte. Using this method, cylindrical nanowires are synthesized, which are released by dissolving the alumina using a selective chemical etchant.

In this work, we describe a&#160;protocol to fabricate iron nanowires, including the formation of the porous alumina membrane that is used as the template, electrodeposition into templates using electrolyte solution, and the release of the nanowires into the solution.

纳米孔阳极氧化铝的铁纳米线制造及其特性

Magnetic nanowires possess unique properties that have attracted the interest of different fields of research, including basic physics, biomedicine, and ...

Organic Structure-directing Agent-free Synthesis for *BEA-type Zeolite Membrane

Membrane separation has drawn attention as a novel-energy saving separation process. Zeolite membranes have great potential for hydrocarbon separation in petroleum and petrochemical fields because of their high thermal, chemical, and mechanical strength. A *BEA-type zeolite is an interesting membrane material because of its large pore size and wide Si/Al range. This manuscript presents a protocol for *BEA membrane preparation by a secondary growth method that does not use an organic structure-directing agent (OSDA). The preparation protocol consists of four steps: pretreatment of support, seed preparation, dip-coating, and membrane crystallization. First, the *BEA seed crystal is prepared by conventional hydrothermal synthesis using OSDA. The synthesized seed crystal is loaded on the outer surface of a 3 cm long tubular &#945;-Al2O3 support by a dip-coating method. The loaded seed layer is prepared with the secondary growth method using a hydrothermal treatment at 393 K for 7 days without using OSDA. A *BEA membrane having very few defects is successfully obtained. The seed preparation and dip-coating steps strongly affect the membrane quality.

A *BEA seed crystal was loaded on a porous &#945;-Al2O3 support by the dip-coating method, and hydrothermally grown without using an organic structure-directing agent. A *BEA-type zeolite membrane having very few defects was successfully prepared by the secondary growth method.

有机结构定向无代理合成，用于_BEA型沸石膜

Membrane separation has drawn attention as a novel-energy saving separation process. Zeolite membranes have great potential for hydrocarbon separation in ...