Carrier design: New generation of polycationic branched polypeptides containing OH groups with prolonged blood survival and diminished in vitro cytotoxicity
F. Hudecz et al., Carrier design: New generation of polycationic branched polypeptides containing OH groups with prolonged blood survival and diminished in vitro cytotoxicity, BIOCONJ CHE, 10(5), 1999, pp. 781-790
For the construction of macromolecule-drug conjugates, it is important to p
rovide rational basis to the selection of proper carrier. With respect to t
he importance of the side-chain structure and charge of the branched polype
ptides in biological properties, we have prepared a new class of branched p
olypeptides with single or multiple hydroxyl groups and studied their solut
ion conformation, in vitro cytotoxicity, biodistribution, and immunoreactiv
ity. For comparative studies, polypeptides were designed to contain serine
at various positions of the side chains, varying also the number. Ser was a
ttached to the end of oligo(DL-Afa) side chains grafted to polylysine resul
ting polypeptides with the general formula poly[Lys(Ser(i)-DL-Ala(m))], (SA
K). Ser was also coupled directly to the polylysine backbone poly[Lys(Ser(i
))] (SiK) and then elongated by polymerization of N-carboxy-DL-Ala anhydrid
e resulting poly[Lys(DL-Ala(m)-Ser(i))] (ASK). An additional polymer was al
so prepared, but instead of the oligo(DL-Ala) branches, oligo(DL-Ser) side
chains were introduced (poly[Lys(DL-Ser(m))], SK). The presence of hydroxyl
groups resulted in compounds with improved of water solubility. CD spectra
of polypeptides showed significant differences correlating with the positi
on and numbers of Ser residues in the side chains. Under physiological cond
itions, polycationic polypeptides assumed ordered secondary structure (SiK
and LSK) or partially unordered conformation (SK, SAK, and ASK). Data of se
lected polymers demonstrate that these polycationic compounds are essential
ly nontoxic in vitro on normal rat liver or mouse spleen cells and have no
cytostatic effect on mouse colorectal carcinoma C26 cells. The blood cleara
nce and biodistribution of these derivatives were greatly dependent on the
position and number of Ser residues in the branches and possess a rather ex
tended blood survival in mice. Polypeptides were taken up predominantly by
the liver and kidney (SiK, LSK, and ASK) or kidney and lung (SK and SAK). T
he best survival in the blood was found with SAK, representing the first po
lycationic branched polypeptide, which show extended blood clearance. The r
elative position of Ser residue had also a marked influence on the immunoge
nicity of polypeptides. The characteristics of the antibody response to pol
ypeptide containing Ser at the end of the branches (SAK) or adjacent to the
polylysine backbone (ASK) was also dependent on the genetic background of
the mouse strains. We also found that these compounds have no effect on to
the SRBC-specific humoral immune response, indicating the lack of nonspecif
ic immunostimulatory potential. In conclusion, these studies suggest that s
ynthetic branched polypeptides with Ser can be considered as candidates for
constructing suitable conjugates for drug/epitope delivery. It is not only
due to the presence of hydroxyl group to be used for oxime chemistry but a
lso to their beneficial biological features.