Surface-Modification of RIPL Peptide-Conjugated Liposomes to Achieve Steric Stabilization and pH Sensitivity.

We have previously demonstrated that RIPL peptide-conjugated liposomes (RIPL-L) exhibited high hepsin (HPN) selectivity and enhanced intracellular drug delivery. In this study, surface modification of RIPL-L was performed to reduce plasma protein adsorption and off-target effects. For steric stabilization, distearoyl phosphatidylethanolamine (DSPE)-polyethylene glycol (PEG)2000 was used (5% molar ratio to total lipid) to prepare PEG-RIPL-L. Further, pH-sensitive oligopeptides [(HD)4 or (HE)4] were coupled to shield the RIPL polyarginine moiety, yielding (HD)4/PEG-RIPL-L and (HE)4/PEG-RIPL-L. All liposomal vesicles had a narrow and homogenous size distribution of approximately 140­150 nm, with zeta potentials varying from −15 to 36 mV. Increased plasma stability was observed upon quantifying the protein adsorbed onto liposomes by using a micro bicinchoninic acid assay. The (HD)4- and (HE)4-coupling capacity of PEG-RIPL-L was investigated by measuring the amount of oligopeptide involved in transient ionic complexation (TIC-oligopep) and zeta potential changes. As the molar ratio of (HD)4 and (HE)4 increased, TIC-oligopep increased and zeta potential decreased. (HE)4/PEG-RIPL-L were pH-sensitive, producing 1.6-fold greater cellular uptake of FITC-dextran by LNCaP cells at pH 6.8 than at pH 7.4. This result suggested that (HE)4/PEG-RIPL-L might provide a sterically stabilized, pH-sensitive drug carrier for HPN-specific cancer targeting.

Design of Multifunctional Liposomal Nanocarriers for Folate Receptor-Specific Intracellular Drug Delivery.

As a novel carrier for folate receptor (FR)-targeted intracellular delivery, we designed two types of targetable liposomal systems using Pep-1 peptide (Pep1) and folic acid as a cell-penetrating peptide (CPP) and target molecule, respectively. Folate-linked Pep1 (Fol-Pep1) was synthesized by solid phase peptide synthesis (SPPS) and verified using (1)H NMR and far-ultraviolet (UV) circular dichroism (CD). The chimeric ligand (Fol-Pep1)-modified liposome (cF-P-L) was prepared by coupling Fol-Pep1 to maleimide-derivatized liposomes at various ratios. The dual ligand (folate and Pep1)-modified liposome (dF/P-L) was prepared by separately attaching both ligands to the liposomal surface via a short (PEG2000) or long (PEG3400) linker. The physical and conformational characteristics including vesicle size, zeta potential, and the number of conjugated ligands were determined. Intracellular uptake specificities of various fluorescent probe-containing cF-P-L and dF/P-L systems were assessed using FR-positive HeLa and FR-negative HaCaT cells. Cellular uptake behavior was visualized by confocal laser scanning microscopy (CLSM). Internalization was time-dependent. Fol-Pep1 and Pep-1 cytotoxicities were negligible up to 25 µM in FR-positive and FR-negative cells. Empty cF-P-L and dF/P-L were nontoxic at the concentration used. The optimized dF3/P2(450/90) system carrying 450 PEG3400-linked folate and 90 PEG2000-linked Pep1 molecules could be a good candidate for FR-specific intracellular drug delivery.

Formulation and in vitro/in vivo evaluation of chitosan-based film forming gel containing ketoprofen.

The film forming gel, adhered to skin surfaces upon application and formed a film, has an advantage onto skin to provide protection and continuous drug release to the application site. This study aimed to prepare a chitosan-based film forming gel containing ketoprofen (CbFG) and to evaluate the CbFG and film from CbFG (CbFG-film). CbFG were prepared with chitosan, lactic acid and various skin permeation enhancers. The physicochemical characteristics were evaluated by texture analysis, viscometry, SEM, DSC, XRD and FT-IR. To identify the mechanism of skin permeation, in vitro skin permeation study was conducted with a Franz diffusion cell and excised SD-rat and hairless mouse dorsal skin. In vivo efficacy assessment in mono-iodoacetate (MIA)-induced rheumatoid arthritis animal model was also conducted. CbFG was successfully prepared and, after applying CbFG to the excised rat dorsal skin, the CbFG-film was also formed well. The physicochemical characteristics of CbFG and CbFG-film could be explained by the grafting of oleic acid onto chitosan in the absence of catalysts. In addition, CbFG containing oleic acid had a higher skin permeation rate in comparison with any other candidate enhancers. The in vivo efficacy study also confirmed significant anti-inflammatory and analgesic effects. Consequently, we report the successful preparation of chitosan-based film forming gel containing ketoprofen with excellent mechanical properties, skin permeation and anti-inflammatory and analgesic effects.