Category: Formulation and Quality
Purpose: Microcapsules obtained from lycopodium (Lycopodium clavatum) spores have been increasingly used as an oral drug and vaccine carrier. A series of chemical treatments involving acetone, KOH, and H3PO4 are used to extract protein-free hollow microcapsules from natural spores. The purpose of this study was to understand the fate of native proteins and wettability of the spores after chemical treatment. Protein-free lycopodium spores are critical to avoid any allergic reaction from intrinsic spore proteins, while amphiphilic spores are critical for formulation development. These aspects of lycopodium spores have not received significant attention.
Methods: Natural lycopodium spores were sequentially treated with acetone, KOH, and H3PO4 to remove proteinaceous materials and to obtain hollow spore shells. Spore surface morphology and structural integrity was analyzed by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and confocal microscopy. To analyze the fate of intrinsic spore proteins after chemical treatment, elemental nitrogen analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) were used. To obtain insight into the functional groups that can affect spore wettability, Fourier transform infrared (FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), and thermogravimetric analysis (TGA) were employed.
Results: Chemical treatment yields a morphologically intact hollow lycopodium microcapsule from natural spores (Figure 1). Natural lycopodium spores are hydrophobic and contain low molecular weight proteins (~ 10 kD) (Figure 2). Acetone treatment partially solubilizes unsaturated phospholipids from the spores. Nevertheless, the acetone-treated spores retain native proteins (Figure 2) and are still hydrophobic. KOH treatment, however, removes a significant amount of proteins (Figure 2) and partially hydrolyzes esters to carboxylic acid salts, and results in a hydrophilic spore with good wettability. Finally, H3PO4 treatment removes residual proteins (Figure 2), hydrolyzes remaining esters to carboxylic acids, and dissolves carbohydrates. H3PO4 treatment temperature controls carbohydrate dissolution, which in turn affects the hydroxyl functional groups and hydrophilicity (wettability) of the treated spores. Spores treated at 60 °C (Figure 3) as opposed to 160 °C are amphiphilic in nature due to the higher abundance of hydroxyl functional groups on the surface.
Conclusion: This study confirms the removal of native proteins from treated lycopodium spores and sheds light on the chemical changes that the spores undergo after chemical treatment. The study also highlights the importance of the temperature of H3PO4 treatment since elevated treatment temperature significantly decreases spore hydrophilicity. Insights gained from this study allowed development of a treatment protocol to obtain protein-free spores that are hydrophylic and can be readily formulated as colloidal suspensions.