Copolymers of poly-L-lysine with serine and tryptophan form stable DNA vectors: implications for receptor-mediated gene transfer.
Inefficient gene transfer and poor stability in physiological medium are important shortcomings for receptor-mediated gene transfer vectors. Here, we evaluate vectors formulated with random copolymers of L-lysine/L-serine (3:1) and L-lysine/L-tryptophan (4:1), focusing on both their biophysical and functional characterization. By means of dynamic light scattering (DLS) and transmission electron microscopy (TEM), we demonstrate that poly-L-lysine (pK), poly-L-lysine-L-tryptophan (pKW) and poly-L-lysine-L-serine (pKS) are able to form compacted, small particles when mixed with plasmid DNA in the absence of salt. Upon dilution in physiological medium, copolymers of both lys/ser and lys/trp do not aggregate, in contrast with poly-L-lysine DNA complexes as determined by scattering, DLS and TEM measurements. Tight packing, as demonstrated by resistance to heparin, SDS and trypsin treatments, is also featured in tryptophan-containing complexes. Successful receptor-mediated endocytosis gene transfer using galactosylated copolymers into cells expressing the asiagloglycoprotein receptor correlated with lack of aggregation. Particles obtained using galactosylated poly-L-lysine-L-tryptophan (Gal-pKW) copolymer demonstrated specific receptor-mediated gene transfer since reporter gene activity dropped in the presence of an excess ligand in the culture medium during transfection. Although copolymers of galactosylated poly-L-lysine-L-serine (Gal-pKS) do not aggregate in the presence of salt, they are not able to internalize in a specific receptor-mediated endocytosis fashion. The introduction of bulky aromatic/hydrophobic (tryptophan) or hydrophillic (serine) moieties into the positively charged vectors allows the compacted particles to disperse into salt-containing medium avoiding salt-induced aggregation. Moreover, tryptophan-containing particles are able to mediate specific gene transfer via receptor-mediated endocytosis.
Gómez-Valadés
A G
AG
Unitat de Biofísica, Departament de Ciències Fisiològiques II, IDIBELL-Universitat de Barcelona, Feixa Llarga s/n, 08907 L'Hospitalet, Spain.
Molas
M
M
Vidal-Alabró
A
A
Bermúdez
J
J
Bartrons
R
R
Perales
J C
JC
D037263:Asialoglycoprotein Receptor / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D045744:Cell Line, Tumor; D004247:DNA / Q000235:genetics* / Q000493:pharmacokinetics; D004355:Drug Stability; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D011107:Polylysine / Q000235:genetics* / Q000493:pharmacokinetics; D011108:Polymers / Q000493:pharmacokinetics; D021381:Protein Transport / Q000235:genetics; D012694:Serine / Q000235:genetics* / Q000493:pharmacokinetics; D014364:Tryptophan / Q000235:genetics* / Q000493:pharmacokinetics
D037263:Asialoglycoprotein Receptor; D011108:Polymers; D011107:Polylysine; D012694:Serine; D014364:Tryptophan; D004247:DNA