Chang Hyun Kim

Surface modification of lipid-based nanocarriers for cancer cell-specific drug targeting

Targeted drug delivery systems using nanocarriers for anticancer drugs have been investigated for over several decades. Among the many nanocarrier systems, lipid-based nanocarriers such as liposomes, solid lipid nanoparticles, and nanostructured lipid carriers have afforded attention as a carrier system to improve the efficacy of anticancer drugs. Recent efforts have focused on cancer cell-specific drug delivery through the functionalization of the surface of lipid-based nanocarriers with various ligands such as targeting moieties, cell-penetrating peptides, and cell-penetrating homing peptides to overcome non-selectivity, minimize side effects, and enhance antitumor efficacy. However, the use of ligand modification has been limited because the nanocarriers were easily recognized by the mononuclear phagocyte system and thus rapidly removed from the blood circulation. To achieve prolonged systemic circulation, nanocarriers were further modified with protective polymers such as polyethylene glycol (PEG). Unexpectedly, this presented a PEG dilemma, as the interaction of ligands with the target was hindered and induced poor cellular uptake. Recently, stimuli-sensitive cleavage of the PEG coat, following recognition of the cancer cell microclimate, such as low pH, redox-potential, and over-expressed enzymes, was established to solve this problem. This review presents a comprehensive overview on the current state of surface-modified lipid-based nanocarriers for the improved delivery of anticancer drugs.

Enhanced Transdermal Delivery by Combined Application of Dissolving Microneedle Patch on Serum-Treated Skin

Dissolving microneedle (DMN), a transdermal drug delivery system in which drugs are encapsulated in a biodegradable polymeric microstructure, is designed to dissolve after skin penetration and release the encapsulated drugs into the body. However, because of limited loading capacity of drugs within microsized structures, only a small dosage can be delivered, which is often insufficient for patients. We propose a novel DMN application that combines topical and DMN application simultaneously to improve skin permeation efficiency. Drugs in pretreated topical formulation and encapsulated drugs in DMN patch are delivered into the skin through microchannels created by DMN application, thus greatly increasing the delivered dose. We used 4-n-butylresorcinol to treat human hyperpigmentation and found that sequential application of serum formulation and DMNs was successful. In skin distribution experiments using Alexa Fluor 488 and 568 dyes as model drugs, we confirmed that the pretreated serum formulation was delivered into the skin through microchannels created by the DMNs. In vitro skin permeation and retention experiments confirmed that this novel combined application delivered more 4-n-butylresorcinol into the skin than traditional DMN-only and serum-only applications. Moreover, this combined application showed a higher efficacy in reducing patients melanin index and hyperpigmented regions compared with the serum-only application. As combined application of DMNs on serum-treated skin can overcome both dose limitations and safety concerns, this novel approach can advance developments in transdermal drug delivery.

Docetaxel-loaded RIPL peptide (IPLVVPLRRRRRRRRC)-conjugated liposomes: Drug release, cytotoxicity, and antitumor efficacy

We previously synthesized the RIPL peptide (IPLVVPLRRRRRRRRC) to facilitate selective delivery into hepsin-expressing cancer cells and showed that RIPL peptide-conjugated liposomes (RIPL-L) enhanced the intracellular delivery of fluorescent probes in vitro. In this study, docetaxel-loaded RIPL-L (DTX-RIPL-L) were prepared and evaluated for in vitro drug release, cytotoxicity, and in vivo antitumor efficacy. DTX was successfully encapsulated by pre-loading, with an average encapsulation efficiency and drug loading capacity of 32.4% and 21.39±2.05 (μg/mg), respectively. A DTX release study using dialysis showed a biphasic release pattern, i.e., rapid release for 6h, followed by sustained release up to 72h. The first-order equation provided the best fit for drug release (r2=0.9349). In vitro cytotoxicity was dose-dependent, resulting in IC50 values of 36.10 (SK-OV-3) and 48.62ng/mL (MCF-7) for hepsin-positive, and 61.12 (DU145) and 53.04ng/mL (PC-3) for hepsin-negative cell lines. Live/dead cell imaging was carried out to visualize the proportion of viable and nonviable SK-OV-3 cells. Compared to DTX solution, DTX-RIPL-L significantly inhibited tumor growth and prolonged survival time in BALB/c nude mice with SK-OV-3 cell tumors. We suggest that DTX-RIPL-L is a good candidate for efficient drug targeting to hepsin-expressing cancer cells.

Development of a chitosan based double layer-coated tablet as a platform for colon-specific drug delivery.

A double layer-coated colon-specific drug delivery system (DL-CDDS) was developed, which consisted of chitosan (CTN) based polymeric subcoating of the core tablet containing citric acid for microclimate acidification, followed by an enteric coating. The polymeric composition ratio of Eudragit E100 and ethyl cellulose and amount of subcoating were optimized using a two-level factorial design method. Drug-release characteristics in terms of dissolution efficiency and controlled-release duration were evaluated in various dissolution media, such as simulated colonic fluid in the presence or absence of CTNase. Microflora activation and a stepwise mechanism for drug release were postulated. Consequently, the optimized DL-CDDS showed drug release in a controlled manner by inhibiting drug release in the stomach and intestine, but releasing the drug gradually in the colon (approximately 40% at 10 hours and 92% at 24 hours in CTNase-supplemented simulated colonic fluid), indicating its feasibility as a novel platform for CDD.

Steric stabilization of RIPL peptide-conjugated liposomes and in vitro assessment

RIPL peptide-conjugated liposomes (PEG-RIPL-Ls) were sterically stabilized with polyethylene glycol (PEG) to prevent recognition by the mononuclear phagocytic system (MPS) and prolong their systemic circulation in vivo. PEG-modified maleimide-derivatized liposomes were prepared by a thin-film hydration method and RIPL peptide was conjugated via a thiol-maleimide reaction. To optimize the system, different chain lengths of PEG were used to prepare PEG-RIPL-L. PEG-RIPL-Ls were positively charged nanodispersions and docetaxel (DTX) was successfully encapsulated by pre-loading with an encapsulation efficiency and drug loading capacity of 31–35% and 15–20 μg/mg, respectively. DTX release showed a biphasic pattern, with rapid release in the initial period of 6xa0h, followed by sustained release for up to 72xa0h. Additionally, 5xa0mol% PEG3000-grafted RIPL-L (PEG3K-RIPL-L) showed enhanced anti-adsorption compared to 5xa0mol% PEG2000-grafted RIPL-L (PEG2K-RIPL-L). Confocal microscopy and flow cytometry using a fluorescence probe (FITC-dextran) demonstrated the greatest stealth effect of PEG3K-RIPL-L. Further analysis of cellular uptake showed that PEG3K-RIPL-L maintained target-selective intracellular delivery capacity. Cytotoxicity analysis demonstrated that PEG3K-RIPL-L had a 1.8-fold lower IC50 value than DTX-Sol. Steric stabilization of RIPL-L was successfully achieved by surface modification with PEG3K, and thus PEG3K-RIPL-L shows potential as a nanocarrier for targeted drug delivery in blood circulation.

RIPL peptide-conjugated nanostructured lipid carriers for enhanced intracellular drug delivery to hepsin-expressing cancer cells

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.

Solid formulation of a supersaturable self-microemulsifying drug delivery system for valsartan with improved dissolution and bioavailability

In order to improve the dissolution and oral bioavailability of valsartan (VST), and reduce the required volume for treatment, we previously formulated a supersaturable self-microemulsifying drug delivery system (SuSMEDDS) composed of VST (80 mg), Capmul® MCM (13.2 mg), Tween® 80 (59.2 mg), Transcutol® P (59.2 mg), and Poloxamer 407 (13.2 mg). In the present study, by using Florite® PS-10 (119.1 mg) and Vivapur® 105 (105.6 mg) as solid carriers, VST-loaded solidified SuSMEDDS (S-SuSMEDDS) granules were successfully developed, which possessed good flow properties and rapid drug dissolution. By introducing croscarmellose sodium (31 mg) as a superdisintegrant, S-SuSMEDDS tablets were also successfully formulated, which showed fast disintegration and high dissolution efficiency. Preparation of granules and tablets was successfully optimized using D-optimal mixture design and 3-level factorial design, respectively, resulting in percentage prediction errors of <10%. In pharmacokinetic studies in rats, the relative bioavailability of the optimized granules was 107% and 222% of values obtained for SuSMEDDS and Diovan® powder, respectively. Therefore, we conclude that novel S-SuSMEDDS formulations offer great potential for developing solid dosage forms of a liquefied formulation such as SuSMEDDS, while improving oral absorption of drugs with poor water solubility.

pH-sensitive PEGylation of RIPL peptide-conjugated nanostructured lipid carriers: design and in vitro evaluation

Background RIPL 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). Methods Various 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. Results All 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. Conclusion cPEG-RIPL-NLCs are good candidates for Hpn-selective drug targeting in conjunction with pH-responsive PEG cleavage.

Formulation and in vivo pharmacokinetic evaluation of ethyl cellulose-coated sustained release multiple-unit system of tacrolimus

A novel once-a-day sustained-release (SR) system of tacrolimus (FK506), a poorly water-soluble immunosuppressive agent, was designed employing ethyl cellulose (EC) polymer as release retardant. Drug (5u202fmg) was layered onto sugar spheres (518.3u202fmg) with hypromellose (5u202fmg), to transform the drug from a crystalline to an amorphous form. Subsequently, the drug-layered pellets were recoated with EC polymer (0.5-1.5u202fmg) using a fluid bed granulator. Drug release from the reservoir-type pellets was markedly impeded by the outer EC-based coating layer (EC 1u202fmg), displaying about 60% of drug release after 8u202fh, regardless of the acidity of the media. In an in vivo pharmacokinetic study in fasted Cynomolgus monkeys, the drug level in blood was gradually increased over 4.7u202fh and high drug concentration was maintained until 24u202fh, with an elimination half-life of 16.6u202fh. There were no statistical differences between the novel SR pellets and the recently marketed SR capsule (Advagraf®, Astellas Pharma, Japan) in terms of maximum blood concentration, area under the curve, and half-life values, in both fasted and fed states. Therefore, the novel EC-coated pellets are expected to be bioequivalent to the commercial SR capsule, providing a once-daily dosing regimen in patients with allogenic rejection.

Tablet Formulation of a Polymeric Solid Dispersion Containing Amorphous Alkalinized Telmisartan

To overcome the poor dissolution of telmisartan (TMS) at weak acidic pH, amorphous alkalinized TMS (AAT) was prepared by introducing sodium hydroxide as a selective alkalizer. AAT-containing polymeric solid dispersions were prepared by a solvent evaporation method; these solid dispersions were AAT-PEG, AAT-PVP, AAT-POL, and AAT-SOL for the polymers of PEG 6000, PVP K30, Poloxamer 407, and Soluplus, respectively. The characteristics of the different formulations were observed by differential scanning calorimetry, powder X-ray diffraction, Fourier transform infrared spectroscopy, and scanning electron microscopy. To compare the supersaturation behavior, a dissolution test was performed at 37u2009±u20090.5xa0°C either in 900xa0ml (plain condition) or 500xa0ml (limited condition) of pH 6.8-simulated intestinal fluid used as a medium. AAT-SOL exhibited enhanced dissolution, indicating the probability of extended supersaturation in the limited condition. AAT-SOL was further formulated into a tablet by introducing other excipients, Vivapur 105 and Croscarmellose, as a binder and superdisintegrant, respectively, using a direct compression method. The selected AAT-SOL tablet was superior to Micardis (the reference product) in the aspect of supersaturation maintenance during dissolution in the limited condition, suggesting that it is a promising candidate for practical development that can replace the commercial product in the future.