Chiral cationic polyamines for chiral microcapsules and siRNA delivery

Article abstract of DOI:10.1016/j.bmcl.2013.08.083

Reported herein is the use of chiral cationic polyamines for two intriguing applications: fabrication of chiral covalently-linked microcapsules, and enantiospecific delivery of siRNA to Huh 7 cells. The microcapsules are easily fabricated from homochiral polymers, and the resulting architectures can be used for supramolecular chiral catalysis and many other potential applications. Enantiospecific delivery of siRNA to Huh 7 cells is seen by one 'enantiomer' of the polymers delivering siRNA with significantly improved transfection efficiency and reduced toxicity compared to the 'enantiomeric' polymer and commercially available transfection reagents. Taken together, the use of these easily accessible polyamine structures for diverse applications is highlighted in this Letter herein and can lead to numerous future research efforts.

Full text of DOI:10.1016/j.bmcl.2013.08.083

Bioorganic & Medicinal Chemistry Letters 23 (2013) 5919–5922
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Chiral cationic polyamines for chiral microcapsules and siRNA
delivery
a
a
a
a
b
b
Justin Gharavi , Patrick Marks , Kelly Moran , Brett Kingsborough , Ruchi Verma , Yuan Chen ,
b
a,
a˜
Ruitang Deng , Mindy Levine
a
Department of Chemistry, University of Rhode Island, 51 Lower College Road, Kingston, RI 02881, United States
College of Pharmacy, University of Rhode Island, 7 Greenhouse Road, Kingston, RI 02881, United States
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Reported herein is the use of chiral cationic polyamines for two intriguing applications: fabrication of chi-
ral covalently-linked microcapsules, and enantiospecific delivery of siRNA to Huh 7 cells. The microcap-
sules are easily fabricated from homochiral polymers, and the resulting architectures can be used for
supramolecular chiral catalysis and many other potential applications. Enantiospecific delivery of siRNA
to Huh 7 cells is seen by one ã enantiomerã of the polymers delivering siRNA with significantly improved
transfection efficiency and reduced toxicity compared to the ã enantiomericã polymer and commercially
available transfection reagents. Taken together, the use of these easily accessible polyamine structures for
diverse applications is highlighted in this Letter herein and can lead to numerous future research efforts.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 25 June 2013
Revised 16 August 2013
Accepted 19 August 2013
Available online 26 August 2013
Keywords:
Polyamine
Chirality
siRNA
Transfection
Polyethyleneimines (PEIs) are a well-studied class of poly-
mers.1 These polymers are synthesized commercially via the ring
The chiral PEIs were synthesized via the cationic polymerization
of 4-benzyl-2-oxazoline (1a) (both R and S configurations), fol-
lowed by the hydrolysis of the initially formed polyformamide
,2
3ã 5
opening of aziridine (Scheme 1, Reaction 2),
although this pro-
cess leads to highly branched polymers6 with significant polydis-
persity indexes. The controlled synthesis of linear PEIs occurs via
the cationic ring opening of oxazolines,7 followed by hydrolysis
of the resulting formamides (Scheme 1, Reaction 1). Using chiral
(Scheme 2). The resulting polymers were characterized by 1
H
NMR spectroscopy, and the results were in agreement with litera-
ã 9
11
ture-reported spectra. Using this methodology, polymers with 13
and 30 repeat units were formed, with both R and S configured side
chains.
oxazolines as substrates for the polymerization reaction provides
straightforward access to homochiral PEIs,1
at every polymer repeat unit.
0ã 12
with chiral centers
Once synthesized, the homochiral PEIs were cross-linked to
form homochiral microcapsules following the procedure devel-
2
2
The significant interest in PEIs is driven largely by various appli-
oped by McQuade and co-workers. Briefly, polymers 2a were dis-
1
3ã 16
cations of PEIs in fields including chiral catalysis,
ery,
drug deliv-
and oligonucleotide complexation and delivery.19,20 PEIs
have also been covalently linked to form PEI-derived microcap-
solved in methanol, and added to a solution of 2% Span 85,
1
7,18
sules,2 which have been used for site-isolated catalysis.
1
22,23
In
one example, the Lewis basic PEI catalyzed a reaction in the same
reaction vessel as a Lewis acidic nickel catalyst, which was used to
catalyze the second reaction.2
4,25
Use of the same PEI scaffold for multiple applications has rarely
been reported, although such multi-purpose polymers would have
significant operational advantages. Reported herein is the use of a
single PEI scaffold for two purposes: the fabrication of covalently-
linked chiral microcapsules, and the efficient delivery of siRNA to
2
6
Huh7 cells.
Scheme 1. General synthetic methods for linear and branched polyethyleneimine
ã
Corresponding author. Tel.: +1 401 874 4243.
E-mail address: mlevine@chm.uri.edu (M. Levine).
(
PEI).
0
960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.08.083

5
920
J. Gharavi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5919–5922
enantioselectivities in such transamination reactions has been an
1
0
ongoing research problem. Preliminary results indicate that the
microcapsule-catalyzed reactions proceeded with significantly
higher enantioselectivities compared to the polymer-catalyzed
reactions (up to 20% enantiomeric excess (ee) obtained for the syn-
thesis of
L-valine, under conditions where the polymer itself
yielded 4% ee).
Scheme 2. Synthesis of chiral polymers 2a.
followed by the addition of 2,4-tolylene diisocyanate (TDI, com-
pound 7) (Equation 1), which cross linked the microcapsules to
form a polyurea coating.25 The resulting polyurethane-type struc-
tures have been shown to be stable in a variety of aqueous
2
7,28
media.
After thorough solvent evaporation, chiral microcap-
sules were obtained.
Efforts to optimize the reaction conditions are in progress.
Interestingly, the chiral PEIs also functioned as efficient siRNA
delivery agents. Although there are many reported examples of
3
1ã 33
PEIs used for siRNA and DNA delivery,
many of these delivery
3
4
vehicles suffer from high cytotoxicity. The development of gene
delivery agents that are both effective and less toxic remains a
highly relevant research objective.
The following 4 polymers were investigated as potential siRNA
delivery agents: R-2a-13; S-2a-13; R-6-13; and S-6-13, where the
R/S designation refers to the chirality of the side chain and the
number 13 refers to the number of repeat units in the polymers.
The efficacy of these polymers in transfecting an Alexa488-labeled
The resulting microcapsules were imaged using transmission
electron microscopy (TEM), and some images are shown in Figure
1
. The diameters of the particles ranged from 57 to 250 nm, with an
35
36
control siRNA sequence to Huh7 cells was measured by deter-
mining the intracellular fluorescence 24 h post-transfection. The
results obtained using the chiral polyamines were compared to re-
sults obtained using commercially available transfection reagents:
Genjet siRNA Transfection Reagent (SignaGen Laboratories); HiPer-
˘
average diameter of 141 nm (ô35 nm; 62 particles measured).
These new supramolecular architectures contain narrow size dis-
tributions and uniform structures, in good agreement with litera-
ture-reported results for achiral microcapsule analogs.21
The newly formed microcapsules contain a variety of features
that make them particularly amenable to supramolecular chiral
catalysis, including: (a) multiple chiral centers, covalently confined
in a small space; (b) multiple amino groups that can be protonated
or deprotonated over a wide pH range;29 and (c) a hydrophobic
core resulting from the hydrophobic benzyl side chains.30
To investigate the effect of capsule formation on the resulting
supramolecular chiral environment, the newly synthesized chiral
microcapsules were used as catalysts for the transamination reac-
tion of ketoacids to amino acids (Equation 2). Obtaining good
37
Fect Transfection Reagent (Qiagen Laboratories); and Lipofect-
38
amine 2000 (Invitrogen Technologies).
Figure 2 shows a graph of the intracellular fluorescence of Huh7
cells following their incubation with Alexa-labeled siRNA with var-
ious delivery reagents. The intracellular fluorescence obtained with
compounds S-6-13 and S-2a-13 is substantially higher than the
fluorescence observed with positive controls Lipofectamine and
Genjet, indicating the polymersã ability to transfect siRNA effi-
ciently (Table 1). More interestingly, compounds R-2a-13 and R-
6-13, which are identical except for the three-dimensional
Figure 1. TEM images of chiral microcapsules 8 (Blue line represents a 500 nm scale).

J. Gharavi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5919–5922
5921
dichroism spectroscopy.4
1ã 43
These differences in chirality affect
4
4
45
the polymersã solubility and their interactions with DNA,
and as shown here, their transfection efficiencies.
In summary, chiral polymers 6 and 2a were synthesized via
straightforward, well-precedented procedures. These polymers
were used for two novel applications: the fabrication of chiral,
covalently-linked microcapsules, and the transfection of siRNA to
Huh7 cells. The chiral microcapsules can be used for a number of
potential applications in supramolecular chiral catalysis and in
supramolecular enantiomer separations. The chirality-dependent
siRNA transfection also provides an intriguing platform for further
investigation. In particular, polymer S-2a-13 demonstrated good
transfection efficiency and limited toxicity, and will be used for
further biochemical investigations. The results of these and other
experiments will be reported in due course.
Acknowledgments
This work was supported by an Institutional Development
Award (IDeA) from the National Institute of General Medical Sci-
ences of the National Institutes of Health under grant number 8
P20 GM103430-12 to M.L. R.D. is supported by the National Insti-
tutes of Health (NIH) (Grant No. R01DK087755).
Figure 2. Chart of the intracellular fluorescence of Huh7 cells after transfection
with siRNA (all PEIs were used at a 1000 nm final concentration).
Table 1
Supplementary data
Transfection efficiencies of chiral PEIs and commercial transfection agents
Transfection agent
Intracellular fluorescence (normalized
to 1.00 for cells alone)
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
S-6-13
1.26
1.30
1.05
1.04
1.06
1.05
0
8.083.
S-2a-13
Lipofectamine
Genjet
R-2a-13
R-6-13
References and notes
1. Jaeger, M.; Schubert, S.; Ochrimenko, S.; Fischer, D.; Schubert, U. S. Chem. Soc.
Rev. 2012, 41, 4755.
2
3
4
5
.
.
.
.
Nimesh, S. Curr. Clinical Pharmacol. 2012, 7, 121.
Kircheis, R.; Wightman, L.; Wagner, E. Adv. Drug Delivery Rev. 2001, 53, 341.
Rivas, B. L.; Geckeler, K. E. Adv. Polym. Sci. 1992, 102, 171.
configuration of the benzyl group, transfect siRNA with approxi-
mately the same efficiency as Lipofectamine and Genjet, and sub-
stantially lower than the ã enantiomericã polymers.
Jones, G. D.; Langsjoen, A.; Neumann, M. M. C.; Zomlefer, J. J. Org. Chem. 1944, 9,
125.
6. Chaikittisilp, W.; Didas, S. A.; Kim, H.-J.; Jones, C. W. Chem. Mater. 2013, 25, 613.
7
8
.
.
Kagiya, T.; Narisawa, S.; Maeda, T.; Fukui, K. J. Polym. Sci. B Polym. Lett. 1966, 4,
41.
Saegusa, T.; Ikeda, H.; Fujii, H. Macromolecules 1972, 5, 108.
The chirality of the side chains of the PEIs thus has a direct and
measurable effect on the ability of PEIs to transfect siRNA effi-
ciently: S chiral centers (compounds S-6-13 and S-2a-13) transfect
siRNA more efficiently than the R analogs. Such a result may seem
intuitive: that the interaction of two chiral macromolecules (chiral
PEI and chiral siRNA) depends on the three-dimensional configura-
tion of both molecules. This intuition is borne out by the results of
this study, which is the first direct proof that the chirality of a poly-
amine directly impacts its transfection efficiency. Similar effects of
the chirality on transfection efficiency were recently observed for
the lipid delivery agent 1,2-dioleoyl-3-trimethylammonium-pro-
4
9. Saegusa, T.; Ikeda, H.; Fujii, H. Polym. J. 1972, 3, 35.
0. Levine, M.; Kenesky, C. S.; Zheng, S.; Quinn, J.; Breslow, R. Tetrahedron Lett.
008, 49, 5746.
1. Bandyopadhyay, S.; Zhou, W.; Breslow, R. Org. Lett. 2007, 9, 1009.
1
2
1
12. Viegas, T. X.; Bentley, M. D.; Harris, J. M.; Fang, Z.; Yoon, K.; Dizman, B.;
Weimer, R.; Mero, A.; Pasut, G.; Veronese, F. M. Bioconjugate Chem. 2011, 22,
976.
1
1
1
3. Zhou, W.; Yerkes, N.; Chruma, J. J.; Liu, L.; Breslow, R. Bioorg. Med. Chem. Lett.
2005, 15, 1351.
4. Kano, T.; Konishi, S.; Shirakawa, S.; Maruoka, K. Tetrahedron Asymmetry 2004,
1
5, 1243.
5. Bellis, E.; Kokotos, G. J. Mol. Catal. A Chem. 2005, 241, 166.
3
9
pane (DOTAP). In that report, the R enantiomer performed better
than either the S enantiomer or the racemic DOTAP mixture.
The toxicity of the newly synthesized PEIs was tested using an
16. Subhani, M. A.; Mueller, K.-S.; Eilbracht, P. Adv. Synth. Catal. 2009, 351, 2113.
17. Kumar, K. S.; Madhusudhanan, J.; Thanigaivel, P.; Robin, A.; Veni, V. A. Res. J.
Biotechnol. 2013, 8, 70.
1
8. Dong, D.-W.; Xiang, B.; Gao, W.; Yang, Z.-Z.; Li, J.-Q.; Qi, X.-R. Biomaterials 2013,
4, 4849.
19. Glebova, K. V.; Marakhonov, A. V.; Baranova, A. V.; Skoblov, M. Y. Mol. Biol.
012, 46, 349.
4
0
MTT assay. After 24 h of incubation, the absorbance of the cells
was quantified and the cell viability was calculated. Using
3
2
1
1
000 nM of S-2a-13 reduced the cell viability to 88%, and
000 nM of S-6-13 reduced it to 82%. By comparison, Lipofect-
2
2
0. Nakayama, Y. Acc. Chem. Res. 2012, 45, 994.
1. Longstreet, A. R.; McQuade, D. T. Acc. Chem. Res. 2013, 46, 327.
amine reduced cell viability to 89%, and compounds R-2a-13 and
R-6-13 reduced viability to 78% and 71%. Thus, the toxicity of the
chiral PEIs, like the transfection efficiency, depends on the three-
dimensional configuration of the benzylic side chains.
The differences in transfection efficiency and toxicity mean that
the S- and R-configured PEIs likely have fundamentally different
three-dimensional architectures. The relationship between the chi-
rality of individual stereocenters and the overall polymer configu-
ration has been studied for related polymers using circular
22. Mason, B. P.; Hira, S. M.; Strouse, G. F.; McQuade, D. T. Org. Lett. 2009, 11, 1479.
2
3. Mason, B. P.; Bogdan, A. R.; Goswami, A.; McQuade, D. T. Org. Lett. 2007, 9,
449.
4. Poe, S. L.; Kobaslija, M.; McQuade, D. T. J. Am. Chem. Soc. 2006, 128, 15586.
3
2
25. Poe, S. L.; Kobaslija, M.; McQuade, D. T. J. Am. Chem. Soc. 2007, 129, 9216.
2
2
6. Ludwig, H. Blood 2010, 116, 3383.
7. Baier, G.; Musyanovych, A.; Dass, M.; Theisinger, S.; Landfester, K.
Biomacromolecules 2010, 11, 960.
28. Lu, Y.; Larock, R. C. Biomacromolecules 2007, 8, 3108.
29. Griffiths, P. C.; Paul, A.; Fallis, I. A.; Wellappili, C.; Murphy, D. M.; Jenkins, R.;
Waters, S. J.; Nilmini, R.; Heenan, R. K.; King, S. M. J. Colloid Interface Sci. 2007,
3
14, 460.

5
922
J. Gharavi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5919–5922
3
3
3
3
3
3
0. Bellettini, I. C.; Nandi, L. G.; Eising, R.; Domingos, J. B.; Machado, V. G.; Minatti,
E. J. Colloid Interface Sci. 2012, 370, 94.
1. Wang, Y.; Su, J.; Cai, W.; Lu, P.; Yuan, L.; Jin, T.; Chen, S.; Sheng, J. Drug Des. Dev.
Ther. 2013, 7, 211.
2. Zhang, Q.-F.; Yi, W.-J.; Wang, B.; Zhang, J.; Ren, L.; Chen, Q.-M.; Guo, L.; Yu, X.-
Q. Biomaterials 2013, 34, 5391.
3. Chu, M.; Dong, C.; Zhu, H.; Cai, X.; Dong, H.; Ren, T.; Su, J.; Li, Y. Polymer Chem.
38. Mo, R. H.; Zaro, J. L.; Ou, J.-H. J.; Shen, W.-C. Mol. Biotechnol. 2012, 51, 1.
39. Terp, M. C.; Bauer, F.; Sugimoto, Y.; Yu, B.; Brueggemeier, R. W.; Lee, L. J.; Lee, R.
J. Int. J. Pharm. 2012, 430, 328.
40. Jiang, G.; Park, K.; Kim, J.; Kim, K. S.; Oh, E. J.; Kang, H.; Han, S.-E.; Oh, Y.-K.;
Park, T. G.; Hahn, S. K. Biopolymers 2008, 89, 635.
41. Luxenhofer, R.; Huber, S.; Hytry, J.; Tong, J.; Kabanov, A. V.; Jordan, R. J. Polym.
Sci., A Polym. Chem. 2013, 51, 732.
42. Bloksma, M. M.; Rogers, S.; Schubert, U. S.; Hoogenboom, R. Soft Matter 2010, 6,
994.
2
013, 4, 2528.
4. Parhamifar, L.; Larsen, A. K.; Hunter, A. C.; Andresen, T. L.; Moghimi, S. M. Soft
Matter 2010, 6, 4001.
5. Alabi, C. A.; Love, K. T.; Sahay, G.; Stutzman, T.; Young, W. T.; Langer, R.;
Anderson, D. G. ACS Nano 2012, 6, 6133.
43. Hoogenboom, R.; Schlaad, H. Polymers 2011, 3, 467.
44. Bloksma, M. M.; Rogers, S.; Schubert, U. S.; Hoogenboom, R. J. Polym. Sci., A
Polym. Chem. 2011, 49, 2790.
3
3
6. Xing, X.-k.; Li, S.-j.; Hafizi, M.; He, J.-l.; Chen, Z. Biotechnol. Lett. 2012, 34, 295.
7. Bakhshandeh, B.; Soleimani, M.; Hafizi, M.; Ghaemi, N. Cytotechnology 2012, 64,
45. Yoshikawa, Y.; Umezawa, N.; Imamura, Y.; Kanbe, T.; Kato, N.; Yoshikawa, K.;
Imanaka, T.; Higuchi, T. Angew. Chem., Int. Ed. 2013, 52, 3712.
5
23.