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accumulation of pDNA (or its complexes) at nucleus periphery after
transfection. For nuclear translocation, due to the high size of the
pDNA molecule, the disassembly of the nuclear envelope must occur
and this will only happen during mitosis. Strategies must, then, be
developed to overcome this difficulty in slowly dividing cells, as is the
case of MSCs. For now, based on the very good capacity of this new
family of gene delivery vectors to internalize nucleic acids, we
hypothesize that these systems may be explored for RNA delivery
(including antisense RNA, small interfering RNA and micro RNA) since
the action site of these molecules is in the cytoplasm. Presently,
ongoing work at our group is devoted to the application of these new
vectors for gene silencing. We are also making further experiments to
understand the mechanism of gene delivery using this family of
vectors.
4. Conclusions
Generation 5 PAMAM dendrimer was successfully functionalized
at its surface with hydrophobic alkyl chains that varied in length and
number. New gene delivery vectors were then synthesized which
showed the ability to neutralize, bind and compact pDNA, as well as to
confer its protection against serum nucleases. Complexes formed
between the vectors and pDNA presented sizes and ζ-potential values
that, respectively, increased and decreased with the length of the
hydrophobic tails. In vitro MSC culture experiments revealed a
remarkable capacity of these vectors for internalizing pDNA with
very low levels of cytotoxicity, being this effect positively correlated
with the –CH2– content present in the hydrophobic moiety. Gene
expression was also enhanced using the new vectors but, in this case,
the higher efficiency was shown by the vectors containing the
smallest hydrophobic chains — La1–G5 and La2–G5 functionalized
dendrimers.
This new family of gene delivery vectors can have important
applications in gene delivery and, thus, in tissue engineering and
regeneration applications where MSCs are often used. Its capacity for
internalizing nucleic acids can also be explored for RNA delivery based
therapies.
Acknowledgements
The Fundação para a Ciência e a Tecnologia (FCT, Portugal) is
acknowledged for funding through the project PTDC/SAU-BEB/71161/
2006 and the Portuguese National Nuclear Magnetic Resonance
Network (PTNMR-REDE/1517/RMN/2005-POCI2010/FEDER), and
also for the Ph.D. scholarship attributed to J. L. Santos (SFRH/BD/
19450/2004). Authors are also grateful to Prof. T. Segura (UCLA, EUA)
for kindly providing the pDNA and to Centro Hospitalar do Funchal for
allowing access to FACS equipment. V. Lather is acknowledged for the
help in the graphical abstract elaboration.
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Appendix A. Supplementary data
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.jconrel.2010.01.034.
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