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BackgroundTo 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.MethodsNLCs 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.ResultsRIPL-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).ConclusionRIPL-NLCs are good candidates for Hpn-selective drug targeting with a high loading capacity of hydrophobic drug molecules.

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BackgroundRIPL peptide (IPLVVPLRRRRRRRRC)-conjugated nanostructured lipid carriers (RIPL-NLCs) can facilitate selective drug delivery to hepsin (Hpn)-expressing cancer cells, but they exhibit low stability in the blood. Generally, biocompatible and nontoxic poly(ethylene glycol) surface modification (PEGylation) can enhance NLC stability, although this may impair drug delivery and NLC clearance. To attain RIPL-NLC steric stabilization without impairing function, pH-sensitive cleavable PEG (cPEG) was grafted onto RIPL-NLCs (cPEG-RIPL-NLCs).MethodsVarious types of NLC formulations including RIPL-NLCs, PEG-RIPL-NLCs, and cPEG-RIPL-NLCs were prepared using the solvent emulsification–evaporation method and characterized for particle size, zeta potential (ZP), and cytotoxicity. The steric stabilization effect was evaluated by plasma protein adsorption and phagocytosis inhibition studies. pH-sensitive cleavage was investigated using the dialysis method under different pH conditions. Employing a fluorescent probe (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate [DiI]), in vitro drug delivery capacity of the cPEG-RIPL-NLCs under different pH conditions was also performed on Hpn-expressing SKOV3 cells and 3D-tumor spheroids.ResultsAll prepared NLCs showed homogenous dispersion (<220 nm in size) with a negative ZP (−18 to −22 mV), except for positively charged RIPL-NLCs (~10 mV), revealing no significant cytotoxicity in either SKOV3 or RAW 264.7 cell lines. cPEG-RIPL-NLC protein adsorption was 1.75-fold less than that of RIPL-NLCs, and PEGylation significantly reduced the macrophage uptake. PEG detachment from the cPEG-RIPL-NLCs was pH-sensitive and time dependent. At 2 hours incubation, cPEG-RIPL-NLCs and PEG-RIPL-NLCs exhibited comparable cellular uptake at pH 7.4, whereas cPEG-RIPL-NLC uptake was increased over 2-fold at pH 6.5. 3D-spheroid penetration also demonstrated pH-sensitivity: at pH 7.4, cPEG-RIPL-NLCs could not penetrate deep into the spheroid core region during 2 hours, whereas at pH 6.5, high fluorescence intensity in the core region was observed for both cPEG-RIPL-NLC-and RIPL-NLC-treated groups.ConclusioncPEG-RIPL-NLCs are good candidates for Hpn-selective drug targeting in conjunction with pH-responsive PEG cleavage.

The objective of this study was to formulate once-a-day extended-release (ER) pellet system of imidafenacin (IDN), a recently approved urinary antispasmodic agent with twice-a-day dosing regimen. The sugar sphere pellets were firstly layered with IDN and hypromellose and then coated with Eudragit RS (copolymers of acrylic and methacrylic acid esters), employed as a release modifier, using a fluid-bed coater. Solid-state characterizations using solid-state X-ray diffraction and differential scanning calorimeter indicated that the antispasmodic agent was homogeneously layered onto the pellets in an amorphous state. Drug release from multiple-unit ER system was effectively retarded in proportion to the amount of Eudragit RS in the outer layer, with a high correlation value above 0.86. In a pharmacokinetic evaluation in beagle dogs, the plasma concentration profile of IDN was markedly protracted by ER pellets, exhibiting delayed the time needed to reach the maximum drug concentration and the elimination half-life in plasma, compared to the commercial immediate release form (Uritos® tablet, Kyorin Pharmaceutical Co., Ltd., Japan). Therefore, the novel ER pellets can be a promising tool for oral IDN therapy, providing a once-a-day dosing regimen, and thus, improving patient compliance.