Kim Beob Soo, Kim Hyun Jin, Osawa Shigehito, Hayashi Kotaro, Toh Kazuko, Naito Mitsuru, Min Hyun Su, Yi Yu, Kwon Ick Chan, Kataoka Kazunori, Miyata Kanjiro
Department of Materials Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology (KIST), Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791, Republic of Korea.
ACS Biomater Sci Eng. 2019 Nov 11;5(11):5770-5780. doi: 10.1021/acsbiomaterials.9b00384. Epub 2019 Aug 15.
For intravenous delivery of antisense oligonucleotides (ASOs) to solid tumors, a triblock copolymer was synthesized from poly(2-ethyl-2-oxazoline) (PEtOx), poly(2--propyl-2-oxazoline) (PnPrOx), and poly(l-lysine) (PLL) segments. The triblock copolymer, PEtOx-PnPrOx-PLL, was utilized to fabricate a compartmentalized polymeric micelle featuring a hydrophilic PEtOx shell, thermoresponsive PnPrOx interlayer, and ASO/PLL polyion complex (PIC) core. In this formulation, the PnPrOx-derived interlayer underwent the phase transition from hydrophilic elongated state to hydrophobic collapsed state at a lower critical solution temperature (LCST) to enhance the micelle stability. Three triblock copolymers comprising varying lengths of PEtOx segment (2k, 7k, and 12 kDa) were compared to investigate the effect of hydrophilic chain length on the micelle properties. The triblock copolymer micelles (TCMs) were prepared in a two-step manner: mixing between triblock copolymer and ASO in a buffer solution at 4 °C and then increasing the temperature of the solution up to 37 °C. This protocol was crucial for the fabrication of TCMs with both smaller size and narrower size distribution, probably due to the formation of the well-compartmentalized hydrophobic interlayer in the micelle structure. The presence of the PnPrOx segment dramatically enhanced the stability of TCMs in serum-containing media and elicited more efficient cellular uptake of ASO payloads, resulting in higher gene silencing efficiency in cultured prostate cancer (PC-3) cells, compared with a control diblock copolymer micelle (DCM). The blood circulation property of TCMs was prolonged with an increase in the length of PEtOx segment, permitting the efficient accumulation of ASO payloads in a subcutaneous PC-3 tumor model. Ultimately, the systemic delivery of ASO targeting a long noncoding RNA (lncRNA) by the TCMs significantly reduced the expression level of lncRNA in the subcutaneous PC-3 tumor in a sequence-specific manner. These results demonstrate the superiority of TCMs equipped with the hydrophilic shell and hydrophobic interlayer to the cancer-targeted systemic ASO delivery.
为了将反义寡核苷酸（ASO）静脉注射至实体瘤，合成了一种由聚（2-乙基-2-恶唑啉）（PEtOx）、聚（2-丙基-2-恶唑啉）（PnPrOx）和聚（L-赖氨酸）（PLL）链段组成的三嵌段共聚物。利用该三嵌段共聚物PEtOx-PnPrOx-PLL制备了一种具有亲水性PEtOx外壳、热响应性PnPrOx中间层和ASO/PLL聚离子复合物（PIC）核心的分隔型聚合物胶束。在此配方中，源自PnPrOx的中间层在较低临界溶液温度（LCST）下经历从亲水性伸长状态到疏水性塌陷状态的相变，以增强胶束稳定性。比较了三种包含不同长度PEtOx链段（2k、7k和12 kDa）的三嵌段共聚物，以研究亲水链长度对胶束性质的影响。三嵌段共聚物胶束（TCM）采用两步法制备：在4℃的缓冲溶液中将三嵌段共聚物与ASO混合，然后将溶液温度升至37℃。该方案对于制备尺寸更小且尺寸分布更窄的TCM至关重要，这可能是由于在胶束结构中形成了分隔良好的疏水中间层。与对照二嵌段共聚物胶束（DCM）相比，PnPrOx链段的存在显著提高了TCM在含血清培养基中的稳定性，并引发了ASO有效载荷更高效的细胞摄取，从而在培养的前列腺癌（PC-3）细胞中产生更高的基因沉默效率。随着PEtOx链段长度的增加，TCM的血液循环特性得以延长，使得ASO有效载荷在皮下PC-3肿瘤模型中有效积累。最终，通过TCM对靶向长链非编码RNA（lncRNA）的ASO进行全身递送，以序列特异性方式显著降低了皮下PC-3肿瘤中lncRNA的表达水平。这些结果证明了配备亲水性外壳和疏水性中间层的TCM在癌症靶向全身ASO递送方面的优越性。
