Marecos E, Weissleder R, Bogdanov A
Center for Molecular Imaging Research, Department of Radiology, Massachusetts General Hospital, Boston 02129, USA.
Bioconjug Chem. 1998 Mar-Apr;9(2):184-91. doi: 10.1021/bc970146w.
The uptake of macromolecular agents in tumor cells (LX-1, human small cell lung carcinoma) and in corresponding tumor xenografts was compared in a parallel study utilizing a long-circulating biocompatible graft copolymer, MPEGs-PL-DTPA [Bogdanov, A., Jr., et al. (1995) Adv. Drug Delivery Rev. 16, 335-348; Bogdanov, A., Jr., et al. (1996) Bioconjugate Chem. 7, 144-149] and a tumor-specific chimeric monoclonal antibody, BR96 [Hellstrom, I., et al. (1990) Cancer Res. 50, 2183-2190; Garrigues, J., et al. (1993) Am. J. Pathol. 142, 607-622]. Covalent grafted conjugates of methoxy-(polyethylene glycol)succinate and polylysine and BR96 were modified with DTPA, biotinyl, or rhodamine-X-residues. Using radionuclide and fluorescent labeled derivatives of the copolymer and the antibody, we established that (1) the copolymer does not associate with the plasma membrane in N-ethylmaleimide-treated cells and is slowly internalized by live cells at 37 degrees C; (2) the antibody binds rapidly to the surface of LX-1 cells and shows active internalization in vesicles with a subsequent slow decrease in the cell-associated antibody concentration; (3) LX-1 cells bear more than 1 million BR96 binding sites/cell (with an apparent Kd of 4.5 x 10[-7] M); and (4) intravesicular fluorescence intensity in LX-1 cells was linearly dependent on copolymer concentration, suggesting fluid phase endocytosis. Tumor localization by nuclear imaging, biodistribution, microdistribution by histology, and determination of tumor cell fraction uptake was performed in LX-1 tumor xenografts. In vivo study showed that MPEGs-PL-DTPA progressively accumulates in the tumor, yielding from 2.8+/-1.5% injected dose per gram of tissue (ID/g) at 24 h to 5.2+/-1.7% ID/g of tissue at 48 h. The antibody accumulation peaked at 24 h (6.0+/-3.2% ID/g) and decreased thereafter. We determined that at 24 h 43.9+/-11.29% of the polymer accumulated in the tumor was associated with tumor cell fraction with the remainder of the accumulated dose localized in the interstitium. Accumulation of the biotinylated graft copolymer and the antibody in LX-1 xenografts and their uptake in cells were confirmed by histology using avidin-peroxidase staining. Our study demonstrates that, although BR96 is highly specific in vitro, tumoral drug delivery in vivo can be equally high with long-circulating graft copolymers because of slow extravasation at the tumor site.
在一项平行研究中，利用一种长循环生物相容性接枝共聚物MPEGs-PL-DTPA [Bogdanov, A., Jr., 等人 (1995) 《药物传递综述进展》16, 335 - 348; Bogdanov, A., Jr., 等人 (1996) 《生物共轭化学》7, 144 - 149] 和一种肿瘤特异性嵌合单克隆抗体BR96 [Hellstrom, I., 等人 (1990) 《癌症研究》50, 2183 - 2190; Garrigues, J., 等人 (1993) 《美国病理学杂志》142, 607 - 622]，比较了大分子药物在肿瘤细胞（LX - 1，人小细胞肺癌）和相应肿瘤异种移植模型中的摄取情况。甲氧基 -（聚乙二醇）琥珀酸酯与聚赖氨酸以及BR96的共价接枝共轭物用DTPA、生物素或罗丹明 - X - 残基进行了修饰。使用共聚物和抗体的放射性核素及荧光标记衍生物，我们确定：(1) 共聚物在N - 乙基马来酰亚胺处理的细胞中不与质膜结合，在37℃时被活细胞缓慢内化；(2) 抗体迅速结合到LX - 1细胞表面，并在囊泡中表现出活跃的内化，随后细胞相关抗体浓度缓慢下降；(3) LX - 1细胞每个细胞带有超过100万个BR96结合位点（表观解离常数Kd为4.5×10[-7] M）；(4) LX - 1细胞内囊泡荧光强度与共聚物浓度呈线性相关，提示液相内吞作用。通过核成像、生物分布、组织学微分布以及肿瘤细胞摄取分数的测定，对LX - 1肿瘤异种移植模型进行了肿瘤定位研究。体内研究表明，MPEGs - PL - DTPA在肿瘤中逐渐积累，从24小时时的每克组织2.8±1.5%注射剂量（ID/g）增加到48小时时的每克组织5.2±1.7% ID/g。抗体积累在24小时达到峰值（6.0±3.2% ID/g），此后下降。我们确定，在24小时时，积累在肿瘤中的聚合物有43.9±11.29%与肿瘤细胞部分相关，其余积累剂量位于间质中。使用抗生物素蛋白 - 过氧化物酶染色的组织学方法证实了生物素化接枝共聚物和抗体在LX - 1异种移植模型中的积累及其在细胞中的摄取。我们的研究表明，尽管BR96在体外具有高度特异性，但由于在肿瘤部位的缓慢渗出，长循环接枝共聚物在体内的肿瘤药物递送效果同样可以很高。
