**Fei Hao 1, Robert J. Lee 1,2, Chunmiao Yang 1, Lihuang Zhong 1, Yating Sun 1, Shiyan Dong 1, Ziyuan Cheng 1, Lirong Teng 1, Qingfan Meng 1, Jiahui Lu 1, Jing Xie 1,\* and Lesheng Teng 1,\***


Received: 24 December 2018; Accepted: 14 February 2019; Published: 21 February 2019

**Abstract:** A combination of chemotherapeutic drugs and siRNA is emerging as a new modality for cancer therapy. A safe and effective carrier platform is needed for combination drug delivery. Here, a functionalized mixed micelle-based delivery system was developed for targeted co-delivery of methotrexate (MTX) and survivin siRNA. Linolenic acid (LA) was separately conjugated to branched polyethlenimine (b-PEI) and methoxy-polyethyleneglycol (mPEG). MTX was then conjugated to LA-modified b-PEI (MTX-bPEI-LA) to form a functionalized polymer-drug conjugate. Functionalized mixed micelles (M-MTX) were obtained by the self-assembly of MTX-bPEI-LA and LA-modified mPEG (mPEG-LA). M-MTX had a narrow particle size distribution and could successfully condense siRNA at an N/P ratio of 16/1. M-MTX/siRNA was selectively taken up by HeLa cells overexpressing the folate receptor (FR) and facilitated the release of the siRNA into the cytoplasm. In vitro, M-MTX/siRNA produced a synergy between MTX and survivin siRNA and markedly suppressed survivin protein expression. In tumor-bearing mice, M-MTX/Cy5-siRNA showed an elevated tumor uptake. In addition, M-MTX/siRNA inhibited tumor growth. Immunohistochemistry and a western blot analysis showed a significant target gene downregulation. In conclusion, M-MTX/siRNA was highly effective as a delivery system and may serve as a model for the targeted co-delivery of therapeutic agents.

**Keywords:** drug co-delivery; methotrexate; siRNA; antitumor effect; mixed micelles; targeted delivery system
