Characterization of Dye-Loaded Poly(lactic- co-glycolic acid) Nanoparticles by Comprehensive Two-Dimensional Liquid Chromatography Combining Hydrodynamic and Reversed-Phase Liquid Chromatography

Abstract

Analytical methods for the assessment of drug-delivery systems (DDSs) are commonly suitable for characterizing individual DDS properties, but do not allow determination of several properties simultaneously. A comprehensive online two-dimensional liquid chromatography (LC × LC) system was developed that is aimed to be capable of characterizing both nanoparticle size and encapsulated cargo over the particle size distribution of a DDS by using one integrated method. Polymeric nanoparticles (NPs) with encapsulated hydrophobic dyes were used as model DDSs. Hydrodynamic chromatography (HDC) was used in the first dimension to separate the intact NPs and to determine the particle size distribution. Fractions from the first dimension were taken comprehensively and disassembled online by the addition of an organic solvent, thereby releasing the encapsulated cargo. Reversed-phase liquid chromatography (RPLC) was used as a second dimension to separate the released dyes. Conditions were optimized to ensure the complete disassembly of the NPs and the dissolution of the dyes during the solvent modulation step. Subsequently, stationary-phase-assisted modulation (SPAM) was applied for trapping and preconcentration of the analytes, thereby minimizing the risk of analyte precipitation or breakthrough. The developed HDC × RPLC method allows for the characterization of encapsulated cargo as a function of intact nanoparticle size and shows potential for the analysis of API stability.

Figure 1

Schematic representation of the HDC × RPLC setup. (A) Intact NP analysis by HDC (¹D), (B) NP disassembly and dilution, (C) SPAM and RPLC analysis (²D) of NP cargo; *, restriction capillary. For disassembly, methods I and II were applied with dilution/disassembly flow rates of 150/250 and 250/150 μL min^–1, respectively.

Figure 2

Nephelometry results of PLGA–PEG-PLGA NPs suspended in mixtures of HDC eluent and ACN. The x-axis depicts the percentage of ACN in the solvent, and the y-axis the observed scatter signal intensity. The means of the 5 technical replicates are plotted as purple points.

Figure 3

Peak areas per modulation as obtained during HDC × RPLC of Sudan-IV. Dilution flow method (A) 250 μL min^–1 and (B) 100 μL min^–1. A scale showing the corresponding NP size is included in the plot.

Figure 4

Normalized peak areas for the dyes obtained with RPLC after online disassembly of the respective NPs as a function of the percentage of ACN that is introduced to the trap columns (i.e., resulting from mixing the ¹D, disassembly, and dilution flow).

Figure 5

Contour plots obtained during HDC × RPLC of dye-loaded NPs. (A) PLGA NP A, (B) PLGA NP B, (C) curcumin-loaded NP (zoom showing 0 to 80 nm range), (D) Sudan-IV-loaded NP. The x-axis depicts the ¹D separation as a function of particle size and the y-axis shows the ²D RPLC separation as a function of retention time.

Figure 6

Mean dye-peak areas per modulation as obtained during HDC × RPLC of dye-loaded NPs. (A) PLGA NP A, (B) PLGA NP B, (C) curcumin-loaded NP, and (D) Sudan-IV-loaded NP. Each sample was measured in triplicate. Error bars indicate the corresponding standard deviations. Scales showing the corresponding NP size are included in the plots.