Biomacromolecules
Article
Horrock’s equation. The water coordination values were found
to be on average slightly less than 1.0, indicating about 1 water
molecule per chelate is directly bound to the metal center.
Because the polymer chelate structure (DTPA) is similar to that
of Magnevist, we expected the water coordination number to
be similar, indicating that the metal chelate likely has a similar
binding stability. Collectively, these data indicate that the
exploration to monitor polyplex dissociation using LRET and
we believe this study opens up avenues to investigate vehicle
unpackaging in cellulo experiments in the future. Current efforts
are directed toward understanding the trend in transfection of
U87 cells, exploring different cell types for the nucleic acid
delivery, evaluating LRET in other nucleic acid types (i.e., RNA),
and exploring in cellulo LRET experiments.
increase in R is primarily due to the larger polymer and polyplex
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structures. These results are promising and promote the further
use and study of these unique systems via MR imaging in the cell
and tissue environment.
ASSOCIATED CONTENT
Supporting Information
Synthesis of monomers, SEC of polymers, DLS data for the
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A primary goal of this study was to develop a lanthanide
resonance energy transfer or LRET-based tool to monitor
polyplex association and dissociation through monitoring the
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formation and dissociation of polyplexes (created with the Tb -
labeled polymers and TMR-labeled pDNA). We found that upon
formation of polyplexes with TMR-labeled pDNA and 6cTb, a
AUTHOR INFORMATION
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significant decrease in emission of Tb luminescence (43%) was
noticed when the emission was monitored at 545 nm at different
N/P ratios of polyplex formulation: 50, 40, 20, 10, and 5.
However, the emission intensity of 6cTb remained unchanged
in polyplexes formulated with unlabeled pDNA, indicating that
the pDNA itself is not a LRET acceptor. The LRET effect was
reversed upon the addition of a NaCl or heparin solution, albeit,
to different degrees. These ions destabilize polyplexes by
weakening the electrostatic interaction between the cationic
polymer and polyanionic pDNA, which causes an increase in
distance between the donor (6cTb) and acceptor (TMR-pDNA)
and can be monitored. During this study, we observed a complete
recovery of Tb3 emission upon addition of heparin (indicating
complete polyplex disruption) and about an 80% recovery with
the addition of NaCl (indicating destabilization) to the polyplex
solution. Thus, we have shown that both polyplex formation
and destabilization/dissociation can be monitored by observing
changes in the emission of Tb (donor) chelated within the
polymer structure. These conditions were meant to mimic the
environment encountered in the cell. Polyplexes are exposed
to high local concentrations of charged macromolecules when
being endocytosed, indicating that competitive binding of
polycations to polyanions within the cell can destabilize
polyplexes and cause nucleic acid release within the cell.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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This project was funded by National Science Foundation (DMR
1105895). The authors would like to thank the following people
for use of instrumentation in their laboratories Prof. Valerie
Pierre, Prof. Webster Santos, Prof. Richey Davis, Prof. Tim Long,
and Prof. Tijana Grove. The authors would also like to thank Drs.
Nilesh Ingle and Giovanna Grandinetti for labeling of the pDNA.
S.K. would like to thank Dr. Karina Kizjakina, Dr. Antons Sizovs,
and Dr. Josh Bryson for helpful discussion with the synthesis.
Authors would also like to thank Techulon Inc. for generous
donation of Glycofect.
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REFERENCES
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+
(1) Sizovs, A.; McLendon, P. M.; Srinivasachari, S.; Reineke, T. M. Top.
Curr. Chem. 2010, 296, 131−190.
(2) Mintzer, M. A.; Simanek, E. E. Chem. Rev. 2009, 109, 259−302.
(
3) Davis, M. E.; Chen, Z.; Shin, D. M. Nat. Rev. Drug Discovery 2008, 7,
7
(
2
(
1
(
1
71−782.
4) Iyer, A. K.; Khaled, G.; Fang, J.; Maeda, H. Drug Discovery Today
006, 11, 812−818.
5) Maeda, H.; Greish, K.; Fang, J. Adv. Polym. Sci. 2006, 193, 103−
21.
6) Kelkar, S. S.; Reineke, T. M. Bioconjugate Chem. 2011, 22, 1879−
903.
(7) Lammers, T.; Aime, S.; Hennink, W. E.; Storm, G.; Kiessling, F.
MTT assays of cultured U87 cells exposed to each of the
polyplex solutions (formed with pDNA and polymers 6a−dTb
and 6a−dGd) at 24 h after transfection indicated no significant
cytotoxicity (∼100% cell survival) with all analogs. Screens of the
cellular transfection efficiency determined using a standard
Luciferase expression assay at 48 h after transfection in U87 cells
revealed that transfection efficiency increased significantly with
the structures containing 6 ethyleneamine units (6d polymers).
However, anomalies with these data were found where 6aGd and
Acc. Chem. Res. 2011, 44, 1029−1038.
(8) Perry, J. L.; Herlihy, K. P.; Napier, M. E.; DeSimone, J. M. Acc.
Chem. Res. 2011, 44, 990−998.
(9) Kunjachan, S.; Jayapaul, J.; Mertens, M. E.; Storm, G.; Kiessling, F.;
Lammers, T. Curr. Pharm. Biotechnol. 2012, 13, 609−622.
(10) Krasia-Christoforou, T.; Georgiou, T. K. J. Mater. Chem. B 2013,
6
bGd structures also showed some transfection, which could be
1
(
(
, 3002−3025.
3
+
related, in part, to the gel shift data that the Gd analogs may
have a slightly stronger binding affinity to pDNA, yet, this effect is
not currently understood.
11) Wang, Z. N.; Gang, C.; Xiaoyuan. Pharm. Res. 2013, 1−19.
12) Ho, Y.-P.; Chen, H. H.; Leong, K. W.; Wang, T.-H. J. Controlled
Release 2006, 116, 83−89.
(13) Shrestha, R.; Elsabahy, M.; Luehmann, H.; Samarajeewa, S.;
CONCLUSION
Florez-Malaver, S.; Lee, N. S.; Welch, M. J.; Liu, Y.; Wooley, K. L. J. Am.
Chem. Soc. 2012, 134, 17362−17365.
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In conclusion, lanthanide resonance energy transfer is a
promising tool to aid understanding of therapeutic packaging,
delivery, and release in vitro and in vivo. Lanthanide labeling
techniques offer many inherent benefits over conventional
organic dyes such as longer luminescence lifetimes, elimination
of background fluorescence with time-gated measurements,
and high signal-to-noise ratios. The current study was our first
(
14) Cutrin, J. C.; Crich, S. G.; Burghelea, D.; Dastru, W.; Aime, S.
Mol. Pharm. 2013, 10, 2079−2085.
15) Chen, H. H.; Ho, Y.-P.; Jiang, X.; Mao, H.-Q.; Wang, T.-H.;
Leong, K. W. Mol. Ther. 2008, 16, 324−332.
16) Cheng, S.-H.; Chen, N.-T.; Wu, C.-Y.; Chung, C.-Y.; Hwu, Y.;
(
(
Mou, C.-Y.; Yang, C.-S.; Lo, L.-W. J. Chin. Chem. Soc. 2011, 58, 798−
804.
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dx.doi.org/10.1021/bm401870z | Biomacromolecules 2014, 15, 1612−1624