Modulation of multidrug resistance by three bisbenzyl-isoquinolines in comparison with verapamil

比较轮环藤碱（Ｃｙｃ）、海岛轮环藤碱（Ｉｎｓｒ）和海岛轮环藤酚碱（Ｉｎｓｎ）与维拉帕米（Ｖｅｒ）体外调节多药耐药性（ＭＤＲ）的作用．方法：细胞毒试验采用ＭＴＴ法，细胞内阿霉素（Ｄｏｘ）积累采用荧光分光光度法测定．结果：Ｃｙｃ，Ｉｎｓｒ，Ｉｎｓｎ和Ｖｅｒ在ＭＤＲ细胞系ＭＣＦ７／Ａｄｒ和ＫＢｖ２００能显著调节Ｄｏｘ和长春新碱的耐药性，且其作用呈剂量依赖性．Ｃｙｃ，Ｉｎｓｒ，Ｉｎｓｎ和Ｖｅｒ均能增加ＭＣＦ７／Ａｄｒ细胞内Ｄｏｘ的积累．Ｃｙｃ和Ｉｎｓｒ调节ＭＤＲ作用明显优于Ｖｅｒ，而Ｉｎｓｎ的作用类似于Ｖｅｒ．结论：Ｃｙｃ，Ｉｎｓｒ和Ｉｎｓｎ能通过增加ＭＤＲ细胞内Ｄｏｘ的积累而调节ＭＤＲ．     

MTT方法测定培养细胞抗药水平的评价

用ＭＴＴ方法测定了多药抗药细胞ＭＣＦ－７Ａｄｒ及其敏感细胞ＭＣＦ－７ＷＴ对阿霉素、长春新碱、秋水仙碱的化学敏感性。ＭＣＦ－７Ａｄｒ与ＭＣＦ－７ＷＴ相比，对三种药物有明显的抗药性。维拉帕米可逆转ＭＣＦ－７Ａｄｒ对阿霉素的抗药性。随选择药物撤除时间的延长，ＭＣＦ－７Ａｄｒ的抗药水平逐渐下降。Ｓｏｕｔｈｅｒｎ杂交显示ＭＣＦ－７Ａｄｒ细胞有典型的ＭＤＲ１基因扩增现象。用ＭＴＴ方法测定Ｓｗｉｓｓ３Ｔ３细胞对阿霉素、长春新碱、秋水仙碱的化学敏感性，同时测定时各亚克隆株间差异较小，而分批测定时，不同批次相差很大。结果说明，ＭＴＴ方法能有效地检测出抗药细胞株的相对抗性；并提示，应在同时用完全相同的条件进行杀伤试验与ＭＴＴ测试，以避免批次间差异。     

吗丙嗪逆转多药抗药性作用及其分子机制的探讨

目的：探讨吗丙嗪逆转肿瘤多药抗药性（ＭＤＲ）的作用及其机制。方法：以ＭＴＴ与Ｆｕｒａ－２－ＡＭ法进行吗丙嗪逆转ＭＤＲ活性测定；以ＤＰＨ荧光测定法探讨吗丙嗪对膜脂流动性的影响；以荧光分光光度计法测定吗丙嗪与高钙或低钙对细胞内阿霉素（Ｄｏｘ）积累的影响及Ｆｕｒａ－２－ＡＭ法测定吗丙嗪对细胞内游离钙离子浓度的影响。结果：在浓度２．５μｍｏｌ·Ｌ－１时，吗丙嗪能显著增加ＭＣＦ－７／ＡＤＲ细胞内Ｆｕｒａ－２的积累和降低ＭＣＦ－７／ＡＤＲ对Ｄｏｘ的ＩＣ５０而对敏感株ＭＣＦ－７无明显影响，表明吗丙嗪具有逆转ＭＤＲ的作用。吗丙嗪能显著增加ＭＤＲ细胞内Ｄｏｘ积累和降低ＭＤＲ细胞的膜脂流动性。高钙或低钙对ＭＤＲ细胞内Ｄｏｘ积累无影响，吗丙嗪也不能改变１９９７－０４－１１收稿１中山医科大学中心实验室，广州５１００８９作者简介：符立梧，男，３２岁，博士，讲师，主要从事逆转多药抗药性的研究；潘启超，男，６７岁，教授，博士生导师，主要从事肿瘤药理与化疗方面的研究ＭＤＲ细胞内游离钙离子浓度。结论：吗丙嗪有逆转ＭＤＲ作用。其逆转机制与降低膜脂流动性增加细胞内ＤＯＸ积累有关，与钙离子浓度无关     

地昔帕明对脑胶质瘤C6细胞的增殖抑制和凋亡诱导作用

目的研究地昔帕明（ＤＭＩ）对大鼠脑胶质瘤Ｃ６的增殖抑制和凋亡诱导作用,为三环类抗抑郁药作为肿瘤辅助治疗提供新的理论和实验依据。方法用ＭＴＴ比色法测定Ｃ６细胞活力,用透射电镜、流式细胞术（ＦＣＭ）检测细胞凋亡,用免疫组化法分析ｂｃｌ－２蛋白的表达。结果ＤＭＩ对Ｃ６细胞的增殖具有明显的浓度依赖性抑制作用, ＩＣ５０（ ９５ ％置信区间）为２０．７（１７．３～２４．２）μｍｏｌ·Ｌ－１,细胞阻滞于Ｇ０～Ｇ１期。ＤＭＩ（４０μｍｏｌ·Ｌ－１）使细胞发生典型的凋亡形态改变,ＤＮＡ含量分析显示 Ｇ０峰左侧出现亚二倍体细胞凋亡峰,呈浓度依赖性,蛋白合成抑制剂ＣＨＸ可显著抑制ＤＭＩ诱导的细胞凋亡。同时,ＤＭＩ（１０ μｍｏｌ·Ｌ－１）可下调细胞 ｂｃｌ－２蛋白的表达。结论ＤＭＩ体外对Ｃ６细胞的增殖具浓度依赖性抑制作用,并诱导细胞凋亡。     

一种新番荔枝内酯单体atemoyacin—B克服肿瘤多药抗药性

目的 探讨ａｔｅｍｏｙａｃｉｎ－Ｂ（Ａｔｅ）克服肿瘤多药抗药性（ＭＤＲ）作用及机制。方法 Ｂｕｌｌａｔａｃｉｎ（Ｂｕｌ）为阳性对照物,细胞毒测定以ＭＴＴ法,Ｐｇｐ功能测定以Ｆｕｒａ２－ＡＭ法,细胞内药物积累测定以荧光分光光度计法;细胞凋亡测定以流式细胞仪法,结果：Ａｔｅ对ＭＣＦ－７／Ｄｏｘ,ＭＣＦ－７,ＫＢＶ２００和ＫＢ细胞的ＩＣ５０分别为１２２,１２０,１．３４,１．２７ｍｍｏｌ．Ｌ＾－１,Ａｔ     

三种双苄基异喹啉生物碱调节多药耐药性的作用及与维拉帕米的比较

目的：比较轮环藤碱（Ｃｙｃ）、海岛轮环藤碱（Ｉｎｓｒ）和海岛轮环藤酚碱（Ｉｎｓｎ）与维拉帕米（Ｖｅｒ）体外调节多药耐药性（ＭＤＲ）的作用。方法：细胞毒试验采用ＭＴＴ法，细胞内阿霉素（Ｄｏｘ）积累采用荧光分光光度法测定。结果：Ｃｙｃ，Ｉｎｓｒ，Ｉｎｓｎ和Ｖｅｒ在分光光度法测定。结果：Ｃｙｃ，Ｉｎｓｒ，Ｉｎｓｎ和Ｖｅｒ在ＭＤＲ细胞系ＭＣＦ－７／Ａｄｒ和ＫＢｖ２００能显著调节Ｄｏｘ和长春新碱的耐药性具有     

番荔枝内酯克服肿瘤多药抗药性作用及机制

目的：探讨阿蒂莫耶番荔枝总内酯（ＡＡ）及其单体ｂｕｌｌａｔａｃｉｎ和ａｔｅｎｏｙａｃｉｎ－Ａ克服肿瘤多药抗药性（ＭＤＲ）的作用及其机制。方法：用两对ＭＤＲ细胞株及其相应的敏感株进行对比研究。结果：ＡＡ及其单体具有极强的细胞毒作用,且对ＭＤＲ细胞与其相应的敏感株的ＩＣ５０相近。加入ＡＡ,ｂｕｌｌａｔａｃｉｎ和ａｔｅｍｏｙａｃｉｎ－Ａ能使细胞内Ｆｕｒａ－２分别增加２．３９,３．１４和３．２４倍,但不能     

粉防己碱对自发性高血压大鼠血管平滑肌细胞增殖及对PDGF-B,bFGF和相关癌基因表达的影响

用［３Ｈ］胸腺嘧啶核苷（［３Ｈ］ＴｄＲ）参入法，电镜，免疫组化，原位杂交方法，在自发性高血压大鼠（ＳＨＲ）观察了粉防己碱（Ｔｅｔ，０．０３μｍｏｌ·ｋｇ－１·ｄ－１×８周ｉｇ）对血管平滑肌细胞（ＶＳＭＣ）增殖的作用及对生长因子ＰＤＧＦ－Ｂ，ｂＦＧＦ的抗原表达及其相关癌基因ｃ－ｓｉｓ，ｃ－ｍｙｃｍＲＮＡ表达的影响．结果发现：Ｔｅｔ在降低ＳＨＲ血压（Ｐ＜０．０１）同时，能减少ＶＳＭＣ的线粒体，粗面内质网和［３Ｈ］ＴｄＲ参入量（Ｐ＜０．０１），并能逆转ＶＳＭＣ增殖时ＰＤＧＦ－Ｂ，ｂＦＧＦ抗原（Ｐ＜０．０５）及ｃ－ｓｉｓ，ｃ－ｍｙｃｍＲＮＡ的表达增强．提示：Ｔｅｔ抑制ＳＨＲ的ＶＳＭＣ增殖与生长因子及癌基因调控的分子生物学机制有关     

Quercetin down-regulated bcl-2 gene expression in human leukemia HL-60 cells

目的：研究槲皮素对人白血病ＨＬ６０细胞ｂｃｌ２基因表达的影响．方法：采用免疫组织化学技术和ＲＮＡ点杂交观察基因表达．结果：Ｑｕｅ１５－６０μｍｏｌ·Ｌ－１处理ＨＬ６０细胞４８ｈ使ｂｃｌ２蛋白由对照组的９４％表达下调到４５％－８４％，并能明显下调ＨＬ６０细胞ｂｃｌ２ｍＲＮＡ表达．结论：Ｑｕｅ诱导ＨＬ６０细胞凋亡，其诱导凋亡的分子机制与下调ｂｃｌ２基因表达有关．     

胃癌组织中细胞凋亡与bcl-2、bax基因蛋白表达的关系

为了探讨胃癌发生发展与细胞凋亡、ｂｃｌ ２ 和ｂａｘ 基因蛋白表达之间的关系,采用原位末端标记ＤＮＡ 断裂片段技术和免疫组化方法对６０ 例胃癌和２５ 例正常胃粘膜中的细胞凋亡指数、ｂｃｌ ２ 和ｂａｘ 进行检测。正常胃粘膜凋亡指数为１１－２ ±４－５１ ,高于胃癌的６－３５ ±３－８４（ Ｐ ＜ ０－０１） 。胃癌中ｂｃｌ ２强表达组凋亡指数为５－１９ ±２－０５ ,低于弱表达组的７－１２ ±３－８９（ Ｐ ＜ ０－０５） ;ｂａｘ 强表达组凋亡指数为７－１９ ±３－７２ ,高于弱表达组的５－２８ ±２－１３（ Ｐ ＜ ０－０５） ,胃癌分化好组凋亡指数为７－８９ ±３－１２ ,高于分化差组的５－２３ ±２－２１（ Ｐ ＜ ０－０５） 。ｂｃｌ ２ 表达越强,ｂａｘ 表达越弱,癌组织分化越差,凋亡指数就越少。提示ｂｃｌ ２ 和ｂａｘ 参与了胃癌组织中细胞凋亡的凋节,并在胃癌的发生发展中发挥着重要作用     

咯萘啶逆转肿瘤多药耐药及其作用机制

目的:利用mdr1~ 的人白血病和乳腺癌多药耐药(MDR)细胞系K562/A02和MCF-7/ADR研究咯萘啶(pyronaridine,PND)对MDR的逆转作用及其机制.方法:采用MTT法、荧光分光光度法、荧光显微镜法、流式细胞仪法和RT-PCR法分别测定PND单独或与阿霉素(DOX)合用,对肿瘤细胞的生长抑制、诱导凋亡、细胞内药物浓度、mdr1基因表达的影响.结果:PND对敏感及耐药细胞均具有生长抑制作用,半数抑制剂量(IC_(50))根据不同细胞在5.10-18.66μmol/L之间;低毒剂量PND显著增强DOX对耐药细胞的细胞毒和诱导凋亡作用,且增加DOX在耐药细胞内的蓄积及减少罗丹明(Rh123)的外排.RT-PCR结果显示,PND对mdr1基因无下调作用.结论:PND可作为第三代P-糖蛋白(P-gp)抑制剂,通过下调P-gp药物外排泵功能而产生强大的逆转MDR效应.     

紫杉醇诱导MCF-7凋亡与bcl-2及bax的关系

目的　探讨抗肿瘤药物紫杉醇及顺铂诱导肿瘤细胞凋亡机制。方法　用紫杉醇及顺铂诱导乳腺癌细胞系MCF 7凋亡 ,采用TUNEL、电镜及免疫组化SP法分别检测凋亡及与凋亡相关基因bcl 2、Bax表达的动态变化。结果　①顺铂仅导致乳腺癌细胞系MCF 7坏死 ,不能诱导其凋亡 ;②紫杉醇可诱导MCF 7细胞凋亡并具有时间及剂量依赖性 ;③bcl 2降低、bax增加具有时间及剂量依赖性。结论　紫杉醇诱导MCF 7细胞凋亡与bcl 2降低及bax增加有关。     

甲基莲心碱对耐阿霉素人乳腺癌细胞凋亡抗性的影响

目的　探讨甲基莲心碱 (neferine ,Nef)对耐阿霉素人乳腺癌细胞 (MCF 7/Adr)凋亡抗性的影响及其机制。方法　应用Tunel法和PI染色流式细胞仪检测细胞凋亡 ,间接免疫荧光流式细胞仪检测P gp的表达 ,高效液相色谱法(HPLC)检测细胞内阿霉素 (ADR)的浓度。结果　①MCF 7/Adr细胞能耐受 5mg·L- 1 ADR诱导的凋亡 ,而 1 ,5 ,1 0μmol·L- 1 Nef可使 5mg·L- 1 ADR诱导的MCF 7/Adr细胞凋亡从 7 95 %分别增加至 2 1 3 % ,2 7 9% ,61 3 % ;② 1 0μmol·L- 1 Nef可使MCF 7/Adr细胞内ADR积累由 0 52 μg·(1 0 6 cells) - 1 增加到 1 50 μg·(1 0 6 cells) - 1 ;③ 1 0 μmol·L- 1Nef作用 2 4h后 ,MCF 7/Adr细胞P gp的表达明显下降。结论　甲基莲心碱能逆转MCF 7/Adr细胞的凋亡抗性 ,其作用机制可能与抑制P gp的功能和表达、增加ADR在MCF 7/Adr细胞内的积累有关     

双苄基异喹啉类生物碱粉防己碱与小檗胺逆转多药抗药性的比较研究

比较了2种结构相近的双苄基异喹啉(BBI)生物碱粉防己碱(TTD)、小檗胺(BBM)与维拉帕米(VRP)逆转多药抗药性的作用。结果,TTD,BBM和VRP在多药抗药的MCF-7/Adr和KBv₂₀₀细胞对ADR和VCR均有明显增敏作用,且作用呈剂量依赖性。其中10μmol·L^-1TTD能完全逆转MCF-7/Adr细胞对ADR的抗药性。TTD,BBM和VRP均有增加MCF-7/Adr细胞内阿霉素积累的作用。TTD和BBM在结构上仅有微小差别,但TTD的逆转MDR作用优于VRP10倍,而BBM的作用与VRP相仿。TTD在裸鼠体内MCF-7/Adr实体瘤模型上也证实有明显逆转ADR抗药性的作用。     

低频超声联合微泡对比剂通过PI3K/AKT通路对乳腺癌耐药株MCF-7/ADR多药耐药的逆转机制研究

目的 探索低频超声联合微泡（LFUSMB）通过磷脂酰肌醇3激酶（PI3K）/蛋白激酶B（AKT）通路对乳腺癌 耐药株MCF-7/阿霉素（ADR）多药耐药的逆转效果及机制。方法 CCK-8法筛选LFUSMB作用时间并测定ADR对 MCF-7/ADR 细胞的半抑制浓度（IC50）。将 MCF-7/ADR 细胞分组为：对照（C）组,采用 MCF 细胞培养基正常培养; ADR组,采用加入ADR（终质量浓度20 mg/L）的MCF细胞培养基培养;LFUSMB+ADR组,采用加入ADR（终质量浓度 20 mg/L）的MCF细胞培养基培养并经LFUSMB处理30 s;LFUSMB+ADR+740 YP（PI3K/AKT通路活化剂）组,采用加 入 ADR（终质量浓度 20 mg/L）和 740 YP（终质量浓度 50 mg/L）的 MCF 细胞培养基培养并经 LFUSMB 处理 30 s。 Annexin V-FIFC/PI 法检测 MCF-7/ADR 细胞凋亡率,Western blot 检测 P 糖蛋白（P-gp）、抗多药耐药蛋白（MRP）1、 MRP2、乳腺癌抗性蛋白（BCRP/ABCG2）、PI3K、AKT、p-PI3K、p-AKT蛋白表达。结果 采用LFUSMB干预30 s进行 后续研究。LFUSMB 30 s组ADR对MCF/ADR细胞的IC50低于未超声处理组,相对耐药逆转倍数约为8.20倍。ADR 组细胞凋亡率较C组增高（P＜0.05）,MRP1、MRP2、P-gp、BCRP/ABCG2、p-PI3K/PI3K和p-AKT/AKT 蛋白水平无明 显变化;LFUSMB+ADR组细胞凋亡率较ADR组增高,MRP1、MRP2、P-gp、BCRP/ABCG2、p-PI3K/PI3K和p-AKT/AKT 蛋白水平显著下调（P＜0.05）。与 LFUSMB+ADR 组比较,LFUSMB+ADR+740 YP 组细胞凋亡率显著降低,MRP1、 MRP2、P-gp、BCRP/ABCG2 蛋白水平显著增高（P＜0.05）。结论 LFUSMB 可通过抑制 PI3K/AKT 通路活化,下调 MRP1等腺苷三磷酸结合盒（ABC）家族蛋白表达,逆转乳腺癌MCF-7/ADR细胞耐药性。     

Effects of miltefosine on various biochemical parameters in a panel of tumor cell lines with different sensitivities

We investigated endocytosis activity, uptake of miltefosine (hexadecylphosphocholine), phospholipid and cholesterol content, the cell cycle, and apoptosis in 13 tumor cell lines (MCF7, MCF7/ADR, KB-3-1, KB-8-5, KB-C1, HeLa, HeLa-MDR1-G185, HeLa-MDR1-V185, CCRF/CEM, CCRF/VCR1000, CCRF/ADR5000, HL-60, HL-60/AR) with different sensitivities to treatment with the antitumor phospholipid analogues miltefosine and D-21266 (octadecyl-(N,N-dimethyl-piperidino-4-yl)-phosphate). In this panel of cell lines, MDR1 (multidrug resistance gene 1)- and MRP1 (multidrug resistance-associated protein)-expressing cells were found to be slightly more resistant to both compounds than sensitive parental cells. No correlation was found between resistance to miltefosine and endocytosis, intracellular concentration of miltefosine, the phospholipid and cholesterol content, induction of apoptosis, or cell cycle alterations in all the cell lines tested. Wild-type p53 containing WMN Burkitt's lymphoma cells and wild type p53-deficient CA46 exhibited similar sensitivities to miltefosine. The low percentage of apoptosis induced in MCF7 cells lacking caspase 3 indicated that caspase 3 seems to play an essential role in miltefosine-induced apoptosis.     

Alteration of genomic responses to doxorubicin and prevention of MDR in breast cancer cells by a polymer excipient: pluronic P85

Polymer therapeutics has emerged as a new clinical option for the treatment of human diseases. However, little is known about pharmacogenetic responses to drugs formulated with polymers. In this study, we demonstrate that a formulation containing the block copolymer Pluronic P85 and antineoplastic drug doxorubicin (Dox) prevents the development of multidrug resistance in the human breast carcinoma cell line, MCF7. Specifically, MCF7 cells cultured in the presence of Pluronic were unable to stably grow in concentrations of Dox that exceeded 10 ng of Dox/mL of culture medium. In sharp contrast, MCF7 cells cultured in the absence of the block copolymer resulted in the selection and stable growth of cells that tolerated a 1000 times higher concentration of the drug (10 000 ng of Dox/mL of culture medium). Detailed characterization of the isolated sublines demonstrated that those cells selected in the polymer-drug formulation did not show amplification of the MDR1 gene, likely resulting in their high sensitivity to the drug. Conversely, cells selected with Dox alone showed an elevated level in the expression of the MDR1 gene along with a corresponding increase in the expression level of the drug efflux transporter, Pgp, and likely contributing to the high resistance of the cells to Dox. Global analysis of the expression profiles of 20K genes by DNA microarray revealed that the use of Pluronic in combination with Dox drastically changed the direction and magnitude of the genetic response of the tumor cells to Dox and may potentially enhance therapeutic outcomes. Overall, this study reinforces the need for a thorough assessment of pharmacogenomic effects of polymer therapeutics.     

Adenovirus-mediated p53 gene therapy reverses resistance of breast cancer cells to adriamycin

The aim of this study was to determine whether adenovirus-mediated p53 gene (Ad-p53) transfection can enhance adriamycin cytotoxicity and reverse adriamycin resistance in human breast cancer cells and explore its effect on the expression of MDR1 gene and permeability-glycoprotein (P-gp). Human breast cancer cell lines, MCF-7 and MCF-7/ADR, were used in in-vitro studies. After infection with Ad-p53, the cytotoxicity of adriamycin was evaluated using the Cell Counting Kit-8 assay. The expression of MDR1 mRNA was detected by quantitative real-time PCR. The expression of P-gp was analyzed using western blotting. In in-vivo studies, MCF-7/ADR tumor cells were inoculated subcutaneously in athymic nude mice. After 14 days of inoculation, tumor size was measured. Apoptosis and expression of P-gp in the tumor tissue were analyzed by fluorescence activated cell sorting and western blotting. After transfection with a multiplicity of infection of 50 for Ad-p53, chemosensitivity of MCF-7/ADR cells increased by 18.1 times (P=0.001), and 50% inhibitory concentration (IC50) of adriamycin decreased from 4.54 ± 0.91 to 0.26 ± 0.11 mg/l. Real-time PCR showed that MDR1 mRNA decreased from 1.32 to 0.85 (P=0.001). Western blotting analysis showed that P-gp also decreased. In in-vivo studies, Ad-p53 combined with adriamycin dramatically inhibited the growth of subcutaneous xenograft of MCF-7/ADR. The fluorescence activated cell sorting assay showed that there were more apoptotic cells in tumor tissues treated with Ad-p53 and adriamycin. The expression of P-gp was significantly decreased in tumor tissues. This study suggests that Ad-p53 can reverse MCF-7/MDR cell resistance to adriamycin. The reversal effect was associated with inhibition of P-gp expression and induction of apoptosis.     

An in vitro demonstration of overcoming drug resistance in SKOV3 TR and MCF7 ADR with targeted delivery of polymer pro-drug conjugates

Drug resistance is a common phenomenon that occurs in cancer chemotherapy. Delivery of chemotherapeutic agents as polymer pro-drug conjugates (PPDCs) pretargeted with bispecific antibodies could circumvent drug resistance in cancer cells. To demonstrate this approach to overcome drug resistance, Paclitaxel (Ptxl)-resistant SKOV3 TR human ovarian- and doxorubicin (Dox)-resistant MCF7 ADR human mammary-carcinoma cell lines were used. Pre-targeting over-expressed biotin or HER2/neu receptors on cancer cells was conducted by biotinylated anti-DTPA or anti-HER2/neu affibody – anti-DTPA Fab bispecific antibody complexes. The targeting PPDCs are either D-Dox-PGA or D-Ptxl-PGA. Cytotoxicity studies demonstrate that the pretargeted approach increases cytotoxicity of Ptxl or Dox in SKOV3 TR or MCF7 ADR resistant cell lines by 5.4 and 27 times, respectively. Epifluorescent microscopy – used to track internalization of D-Dox-PGA and Dox in MCF7 ADR cells – shows that the pretargeted delivery of D-Dox-PGA resulted in a 2- to 4-fold increase in intracellular Dox concentration relative to treatment with free Dox. The mechanism of internalization of PPDCs is consistent with endocytosis. Enhanced drug delivery and intracellular retention following pretargeted delivery of PPDCs resulted in greater tumor cell toxicity in the current in vitro studies.