Characterization - Lipid Nanoparticles: Production, Characterization and Stability - page 50

Fig. 4.2 Freeze-fracture
TEM of nanostructured lipid
carriers prepared by high
pressure homogenization.
(Composition: 5 % octyl
decyl acid triglyceride,
5 % soyabean lecithin,
5 % Coenzyme Q10 and
45 % glycerol). Reprinted
from Int J Pharm, Yue et al.
( 2010 ), with permission from
Elsevier
gold or copper. The drop of dispersion is vitrified by rapid freezing in a cryogen
such as liquid propane or liquid nitrogen. The frozen sample is fractured under
constant cooling and high-vacuum. The fracture plane develops at the sites with
low binding forces. The fracture plane may further be etched by sublimation of
the frozen water under vacuum. The sample surface shadowed with a thin carbon/
platinum film at a 45° angle provides a negative replica of the fractured plane. The
thin film is stabilized with a thicker carbon film (~30 nm). The organic residues
are removed by cleaning the replica with an organic solvent and viewing the rep-
lica by TEM (Severs 2007 ). Figure 4.2 is an illustrative example of NLCs prepared
by the freeze-fracture technique.
Freeze-fracture transmission electron micrographs are very helpful in elucidat-
ing the size, shape and internal structure of colloidal carriers. This technique has
been used to distinguish between different colloidal structures such as solid lipid
particles, emulsions and liposomes (Westesen et al. 2001 ). The influence of pol-
ymorphic transitions on particle structure and morphology has also been shown
with this technique (Bunjes et al. 2003 ). The freeze-fracture TEM technique was
used to visualize the changes in particle morphology due to intraparticulate phase
separation between the lipid matrix and incorporated drug molecules (Bunjes
et al. 2001 ).
The freeze-fracture technique has also been employed to investigate the for-
mation of gels in phospholipid-stabilized lipid dispersions, stacking of particles
in concentrated suspensions and localization in semi-solid preparations (Unruh
et al. 1999 ). Although freeze-fracture has been widely used in elucidating the