4. The MOI describes the number of virus particles used to infect
one cell. The probability of infecting a cell, however, is subject
to the statistical Poisson distribution. For example, an MOI of
100 indicates that 100 virus particles are needed for one cell to
be infected efficiently. In principle, the MOI for Ad particles
ranges from 10 to 1000, depending on the particular cell line.
5. Stability of NHS esters are compared by their hydrolysis halflife (t 1/2 ). The hydrolysis half-life of the PEG-SCM is 1–5 min.
Thus, dissolve PEG in buffer just before adding to the virus.
Avoid frequent freeze/thaw cycles.
6. A 1 ml stock solution (1 mg/ml) of MAL-PEG-NHS and an
800 μl of stock solution (2 mg/ml) of CPP will be sufficient to
prepare enough PEN-PEG-Ad solution to transduce a 24-well
plate.
7. As PEG molecules conjugate to the surface of the virus, they
mask protein residues that decrease the overall charge while
increasing the particle size of Ad [21, 22]. Thus, initial characterization of PEGylated adenoviruses is to assess the physicochemical properties of the virus such as particle size and zeta
potential. The amount of PEG conjugated to Ad is estimated
using a fluorescamine assay [23], in which fluorescamine reacts
with free lysine on the virus capsid to estimate the degree of
PEGylation (DOP). Briefly, 50 μl of fluorescamine (Sigma,
St. Louis, MO), at a concentration of 0.6 mg/ml in acetone,
is added to serial dilutions of modified and unmodified virus
samples at room temperature and left for 15 min. Fluorescence
measurements are then measured using a PTI fluorometer
(Photon Technologies International, Edison, NJ), with an
excitation wavelength of 390 nm and an emission wavelength
of 475 nm. Fluorescence measurements are plotted against
virus concentration, and the amount of PEG conjugated
is determined by comparing the slopes of the modified to the
unmodified virus sample [20].
8. An Ellman’s assay is used to quantify the amount of free thiol,
corresponding to unreacted cysteine residues of CPPs that are
not conjugated to the PEG-Ad particles [19]. The Ellman’s
reagent, 5,5
0 -dithio-bis-2-nitrobenzoic acid (DTNB), has an
oxidizing disulfide bond that is reduced in the presence of
free thiols forming a mixed disulfide and releasing one molecule of 5-thio-2-nitrobenzoic acid (TNB). TNB is a yellow
product that can be quantified by measuring absorbance at
412 nm. The amount of free CPP in the sample is estimated
by comparing the sample absorbance to a standard curve composed of known concentrations of a sulfhydryl-containing
compound (DNTB thiol).
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Yasmine Gabal and Joshua D. Ramsey
one cell. The probability of infecting a cell, however, is subject
to the statistical Poisson distribution. For example, an MOI of
100 indicates that 100 virus particles are needed for one cell to
be infected efficiently. In principle, the MOI for Ad particles
ranges from 10 to 1000, depending on the particular cell line.
5. Stability of NHS esters are compared by their hydrolysis halflife (t 1/2 ). The hydrolysis half-life of the PEG-SCM is 1–5 min.
Thus, dissolve PEG in buffer just before adding to the virus.
Avoid frequent freeze/thaw cycles.
6. A 1 ml stock solution (1 mg/ml) of MAL-PEG-NHS and an
800 μl of stock solution (2 mg/ml) of CPP will be sufficient to
prepare enough PEN-PEG-Ad solution to transduce a 24-well
plate.
7. As PEG molecules conjugate to the surface of the virus, they
mask protein residues that decrease the overall charge while
increasing the particle size of Ad [21, 22]. Thus, initial characterization of PEGylated adenoviruses is to assess the physicochemical properties of the virus such as particle size and zeta
potential. The amount of PEG conjugated to Ad is estimated
using a fluorescamine assay [23], in which fluorescamine reacts
with free lysine on the virus capsid to estimate the degree of
PEGylation (DOP). Briefly, 50 μl of fluorescamine (Sigma,
St. Louis, MO), at a concentration of 0.6 mg/ml in acetone,
is added to serial dilutions of modified and unmodified virus
samples at room temperature and left for 15 min. Fluorescence
measurements are then measured using a PTI fluorometer
(Photon Technologies International, Edison, NJ), with an
excitation wavelength of 390 nm and an emission wavelength
of 475 nm. Fluorescence measurements are plotted against
virus concentration, and the amount of PEG conjugated
is determined by comparing the slopes of the modified to the
unmodified virus sample [20].
8. An Ellman’s assay is used to quantify the amount of free thiol,
corresponding to unreacted cysteine residues of CPPs that are
not conjugated to the PEG-Ad particles [19]. The Ellman’s
reagent, 5,5
0 -dithio-bis-2-nitrobenzoic acid (DTNB), has an
oxidizing disulfide bond that is reduced in the presence of
free thiols forming a mixed disulfide and releasing one molecule of 5-thio-2-nitrobenzoic acid (TNB). TNB is a yellow
product that can be quantified by measuring absorbance at
412 nm. The amount of free CPP in the sample is estimated
by comparing the sample absorbance to a standard curve composed of known concentrations of a sulfhydryl-containing
compound (DNTB thiol).
364
Yasmine Gabal and Joshua D. Ramsey