Profiling in-vitro release of verteporfin from VISUDYNE® liposomal formulation and investigating verteporfin binding to human serum albumin.

VISUDYNE® is a liposomal formulation of verteporfin, used in the photodynamic therapy of age-related macular degeneration via intravenous administration. In this study, we developed a new in vitro method to quantify verteporfin release from VISUDYNE® under conditions that replicate in vivo conditions using human serum albumin (HSA). Verteporfin release from the liposomes was quantified using capillary electrophoresis (CE) with optical detection. Verteporfin binding to HSA was quantified by measuring HSA fluorescence that is quenched by drugs binding to specific HSA binding sites. The binding constant of verteporfin to HSA was calculated using the Stern Volmer plot and found to be 1.966 × 107 M-1 at 37 °C. Verteporfin binding to HSA involves one albumin binding site and the binding molar ratio between verteporfin and HSA is approximately 1:1. A rapid partitioning of verteporfin from VISUDYNE® onto HSA takes place within 10 min and involves the release of more than 90% of the verteporfin at physiological temperatures. This study verifies this approach of using CE to rapidly separate liposome and HSA-bound drug, thus minimizing drug release artifacts created with other methods.

Quantification of phospholipid degradation products in liposomal pharmaceutical formulations by ultra performance liquid chromatography-mass spectrometry (UPLC-MS).

Identification and quantification of excipient related degradation products in the liposomal formulation is important, as they may impact the safety and efficacy of the drug. Phospholipids are one of the major excipients in liposome drugs composing the lipid bilayer, and they are vulnerable to oxidation and hydrolysis reactions. Since phospholipids with saturated fatty acid chain were preferred in most of liposome drug products, the major degradation pathway of phospholipids in liposome formulations are limited to hydrolysis of phospholipids into free fatty acids and lysophospholipids. These hydrolyzed degradation products may form during manufacturing and/or long-term storage of liposomal formulations. Herein, we report development and application of accurate and sensitive methods that can be utilized for the quantitation of saturated free fatty acids (FFA 18:0 and FFA 16:0), lysophosphocholines (LPC 18:0 and LPC 16:0), and lysophosphoglycerol (LPG 18:0) in liposomal formulations. The free fatty acids were separated using a C8 column whereas the LPCs and LPGs were separated using a C18 stationary phase upon direct injection without the need of lipid extraction process. Each analyte was quantified by Q-TOF mass spectrometry. This method was validated according to USP compendial procedures and has been applied to the analysis of four commercial liposomal pharmaceutical formulations. The limit of quantitation (LOQs) of FFA 16:0, FFA 18:0, LPC 16:0, LPC 18:0 and LPG 18:0 are 5 ng/mL, 5 ng/mL, 6.5 ng/mL, 7.0 ng/mL, 10 ng/mL respectively. Compared to CAD (Charge Aerosol Detector) and ELSD (Evaporative Light Scattering Detector) detection methods in ppm levels, this ultra-performance liquid chromatography (UPLC)-Mass Spectroscopy (MS) method displays precise determination of lysophospholipids in the liposomal formulations with higher accuracy and sensitivity.

Quantitative analysis of cholesterol oxidation products and desmosterol in parenteral liposomal pharmaceutical formulations.

Cholesterol is one of the major structural constituents in a liposomal bilayer. Cholesterol is susceptible to various reactions in the presence of oxygen, heat, light, certain metals, and radicals during manufacturing or storage, which may cause to generate cholesterol oxidation products (COPs). Herein, we report the development of a liquid chromatography-mass spectrometry based analytical method for screening and quantitating COPs present in liposomal parenteral pharmaceutical formulations (LPFs) from four different vendors. We detected and quantitated six COPs and desmosterol in LPFs, and desmosterol is an intermediate of cholesterol biosynthesis. 7α-hydroxycholesterol, 7ß-hydroxycholesterol, 7-keto-cholesterol, and desmosterol were the major cholesterol-related impurities in LPFs. COPs were not detected in any of USP/NF grade cholesterol raw materials, implying that COPs were generated during liposome manufacturing and/or storage. This validated method presented here can be used to quantify cholesterol-related impurities present in liposomal pharmaceutical formulations to ensure the quality and the safety of liposomal pharmaceutical formulations.

Self-assembling Morphologies Obtained from Helical Polycarbodiimide Copolymers and Their Triazole Derivatives.

A facile method for the preparation of polycarbodiimide-based secondary structures (e.g., nano-rings, "craters," fibers, looped fibers, fibrous networks, ribbons, worm-like aggregates, toroidal structures, and spherical particles) is described. These aggregates are morphologically influenced by extensive hydrophobic side chain-side chain interactions of the singular polycarbodiimide strands, as inferred by atomic force microscopy (AFM) and scanning electron microscopy (SEM) techniques. Polycarbodiimide-g-polystyrene copolymers (PS-PCDs) were prepared by a combination of synthetic methods, including coordination-insertion polymerization, copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) "click" chemistry, and atom transfer radical polymerization (ATRP). PS-PCDs were found to form specific toroidal architectures at low concentrations in CHCl3. To determine the influence of a more polar solvent medium (i.e., THF and THF/EtOH) on polymer aggregation behavior, a number of representative PS-PCD composites have been tested to show discrete concentration-dependent spherical particles. These fundamental studies are of practical interest to the development of experimental procedures for desirable architectures by directed self-assembly in thin film. These architectures may be exploited as drug carriers, whereas other morphological findings represent certain interest in the area of novel functional materials.