| Summary: | Sang Gon Lee,1,* Chang Hyun Kim,1,* Si Woo Sung,1 Eun Seok Lee,1 Min Su Goh,1 Ho Yub Yoon,1 Myung Joo Kang,2 Sangkil Lee,3 Young Wook Choi1 1College of Pharmacy, Chung-Ang University, Seoul, Republic of Korea; 2College of Pharmacy, Dankook University, Chungnam, Republic of Korea; 3College of Pharmacy, Keimyung University, Daegu, Republic of Korea *These authors contributed equally to this work Background: To facilitate selective and enhanced drug delivery to hepsin (Hpn)-expressing cancer cells, RIPL peptide (IPLVVPLRRRRRRRRC, 16-mer)-conjugated nanostructured lipid carriers (RIPL-NLCs) were developed. Methods: NLCs were prepared using a solvent emulsification-evaporation method and the RIPL peptide was conjugated to the maleimide-derivatized NLCs via the thiol-maleimide reaction. Employing a fluorescent probe (DiI), in vitro target-selective intracellular uptake behaviors were observed using fluorescence microscopy and flow cytometry. Separately, docetaxel (DTX) was encapsulated by pre-loading technique, then cytotoxicity and drug release were evaluated. In vivo antitumor efficacy was investigated in BALB/c nude mice with SKOV3 cell tumors after intratumoral injections of different DTX formulations at a dose equivalent to 10 mg/kg DTX. Results: RIPL-NLCs showed positively charged nanodispersion, whereas NLCs were negatively charged. DTX was successfully encapsulated with an encapsulation efficiency and drug loading capacity of 95–98% and 44-46 µg/mg, respectively. DTX release was diffusion-controlled, revealing the best fit to the Higuchi equation. Cellular uptake of DiI-loaded RIPL-NLCs was 8.3- and 6.2-fold higher than that of DiI-loaded NLCs, in Hpn(+) SKOV3 and LNCaP cells, respectively. The translocation of RIPL-NLCs into SKOV3 cells was time-dependent with internalization within 1 h and distribution throughout the cytoplasm after 2 h. DTX-loaded RIPL-NLCs (DTX-RIPL-NLCs) revealed dose-dependent in vitro cytotoxicity, while drug-free formulations were non-cytotoxic. In SKOV3-bearing xenograft mouse model, DTX-RIPL-NLCs significantly inhibited tumor growth: the inhibition ratios of the DTX solution-treated and DTX-RIPL-NLC-treated groups were 61.4% and 91.2%, respectively, compared to those of the saline-treated group (control). Conclusion: RIPL-NLCs are good candidates for Hpn-selective drug targeting with a high loading capacity of hydrophobic drug molecules. Keywords: nanostructured lipid carriers, RIPL peptide, intracellular delivery, docetaxel, antitumor efficacy, targeting
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