crc press - E-Lib FK UWKS

334 Cell-Penetrating Peptides: Processes and Applications
conjugation with specific groups on cargo molecules and CPPs. Using heterobifunctional
reagents, the most labile group should be reacted first to ensure effective crosslinking
and avoid unwanted polymerization. The second reaction can then be initiated
after cleaning the excess reagents and byproducts from the first. Linker selection is
based on the reactive group on either molecule. The majority of commercially
available heterobifunctional linkers contain an amine-reactive at one end and a thiolreactive
at the other.
If the CPP were modified to have a thiol group as previously described, the
first step of the conjugation usually begins by reacting the activated ester with the
amino group on the cargo molecules at basic pH. Then the CPP is attached to the
activated first component through a thiol-specific reaction. The commonly used
linkers include m-Maleimidobenzoyl-N-hydroxysuccinimide (MBS), N-succinimidyl
3-(2-pyridyldithio)propionate (SPDP), iodoacetic anhydride, and other similar analogs.
Here we use an example to illustrate the solution conjugation approach. A
magnetic particle and the Tat peptide were linked using SPDP. The described protocol
can also be applied in CPP and protein conjugation.
EXAMPLE 15.2
PREPARATION OF SUPERPARAMAGNETIC IRON OXIDE PARTICLE AND TAT PEPTIDE
CONJUGATE (FIGURE 15.4). 34,35
1. Reagents:
Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Gly-Tyr-Lys(FITC)-Cys-NH 2
(the italicized amino acids correspond to CPP signal of the Tat protein)
N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (Molecular Biosciences,
Boulder, CO)
2. An amine terminated cross-linked monodispersed superparamagnetic iron
oxide colloid (CLIO-NH 2) was prepared as described. 35 The particles
consist of a small monocrystalline, superparamagnetic iron oxide core,
stabilized by a cross-linked dextran coating to improve stability that was
aminated. The particle core measures approximately 5 nm and the overall
particle size is 45 nm as determined.
3. CLIO-NH 2 (31.2 mg Fe, 557 µmol) in 2.4 mL of 0.1 M phosphate buffer,
pH 7.4 was added 2 mL of SPDP in DMSO (25 mM, 50 µmol). The
reaction mixture was allowed to stand for 3 h at room temperature.
4. Low molecular impurities such as excess reagent and NHS were removed
by a gel filtration Sephadex G-25 columns (20 cm, Sigma Chemical, St.
Louis, MO) equilibrated with 0.01 M Tris, 0.02 M citrate, pH 7.4 buffer.
The pooled void volume of 4.4 mL was recovered, containing 2-pyridyl
disulfide derivatized CLIO at a concentration of 7 mg Fe/mL (0.125 M).
5. To measure the number of 2-pyridyl disulfide groups attached, 0.2 mL of
the solution above was added to 0.2 mL of 50 mM DTT in 0.1 M phosphate,
pH 7.4. The mixture was allowed to stand for 30 min at room
temperature. A microconcentrator, 30 kDa cutoff (Amicon, Beverly, MA),
was used to separate the product of the reduction, pyridine-2-thione, from
iron. The concentration of pyridine-2-thione was quantitated using an