Nanoparticles for Drug and Gene Delivery in Treating Diseases of the Eye - Ocular Pharmacology and Toxicology - page 295

5. Following staining in step 4, the grid is blotted with a filter
paper and air dried for 5 min.
6. The sample is examined under an electron microscope set at
60-100 kV ( see Note 36 ).

Essentially, the same sample preparation used for particle size deter-
mination can be used for zeta measurements. If the sample is at the
right dilution for size measurements, but is too concentrated or
dilute for zeta potential measurements, a fresh dispersion suitable
for zeta potential measurements should be prepared in such cases.
Because zeta potential measures the potential difference between
the dispersion medium and the stationary layer of liquid attached to
the dispersed particle, the dispersion medium in which the sample is
prepared can affect the zeta potential measurement. Different media
that are commonly employed are de-ionized water, PBS, and saline.
Selection of the medium would depend on the conditions in which
the zeta potential is desired. If the actual zeta potential of the
particles is desired without any influence of the dispersion medium
on the particle charge, de-ionized water would be the ideal choice.
However, if the zeta potential of a particle in a physiological medium
such as serum is desired, then the measurement should be done in
PBS. Zeta potential measurements can be done using the Zetasizer
Nano ZS (Malvern ® Instruments) and are based on the principle of
laser doppler micro-electrophoresis. Zeta potential measurements
require the disposable folded capillary cells, which are specially
designed for measuring zeta potential ( see Note 37 ). PLGA nano-
particles prepared by the methods described are reported to have a
negative zeta potential in water [ 7 , 19 ].

3.2.3 Zeta Potential

Drug loading is an estimate of the amount of drug entrapped in the
nanoparticles and can be used for calculating the entrapment effi-
ciency of the particles.

3.2.4 Drug Loading

Mass of the drug in nanoparticles
Mass of nanoparticles weighed

Percent drug loading ¼ 100

Experimental drug loading
Theoretical drug loading 100
Drug loading can be quantified by following the steps
described below:

Percent entrapment efficiency ¼

1. 2 mg of the lyophilized nanoparticles are weighed ( see

Note 38 ).
2. The weighed nanoparticles are dissolved in 2 ml DCM (or
chloroform) and vortexed for about 1 h at room temperature
( see Note 39 ).