TACN-based oligomers with aromatic backbones for efficient nucleic acid delivery

Article abstract of DOI:10.1039/c4cc01210k

Cationic oligomers with a rigid aromatic backbone were first applied as non-viral gene delivery vectors. These materials showed better DNA condensation ability than their flexible analogues. In vitro transfection experiments revealed that the materials with more rigid backbone exhibited considerably higher TE and lower cytotoxicity than 25 kDa PEI. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc01210k

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TACN-based oligomers with aromatic backbones
for efficient nucleic acid delivery†
Cite this: Chem. Commun., 2014,
50, 6454
Wen-Jing Yi,a Xing-Chi Yu,a Bing Wang,a Ji Zhang,*a Qing-Ying Yu,a
Xue-Dong Zhou*b and Xiao-Qi Yu*a
Received 16th February 2014,
Accepted 27th March 2014
DOI: 10.1039/c4cc01210k
www.rsc.org/chemcomm
Cationic oligomers with a rigid aromatic backbone were first applied binding and gene transfection.12 So far, most current hydrophobic
as non-viral gene delivery vectors. These materials showed better modification studies intensively aim at conjugation with aliphatic
DNA condensation ability than their flexible analogues. In vitro alkyl groups that vary in length and number.13 Nevertheless, the
transfection experiments revealed that the materials with more rigid effect of alkyl modification on the transfection is still controversial.14
backbone exhibited considerably higher TE and lower cytotoxicity Although some more rigid moieties such as aromatic rings,15
than 25 kDa PEI.
cholesterol,16 folate,17 and glucocorticoid ligand18 have been
attached to polycations, polymeric gene vectors with aromatic
Design of safe and efficient gene vectors is currently an urgent backbones have been seldom studied.
challenge for gene therapy. In the past decades, various cationic
Our group recently developed non-viral gene vectors with the
polymers have emerged as one type of promising non-viral gene structures based on 1,4,7-triazacyclononane (TACN) due to its
vector candidates.1 In contrast to viral vectors, polycations have low unique characteristics, especially pH buffering capacity.19
immunogenicity and high nucleic acid loading capacity, and they Herein, we report a new type of TACN-based oligomers with
can be prepared reproducibly, designable, inexpensively, and in aromatic phenyl groups in the backbone (Fig. 1). Results
large-scale.2 Cationic polymers can electrostatically compact nucleic revealed that the aromatic moiety plays an important role in
acids into nanoparticles termed ‘‘polyplexes’’.3 The condensation the gene transfection process, and much higher TE and lower
may facilitate cellular uptake through various endocytic pathways, cytotoxicity were obtained by comparison with the ‘‘golden
and inhibit intra- or extracellular enzymatic degradation.4 Common standard’’ 25 kDa branched PEI.
cationic polymers such as polyethyleneimine (PEI),5 Superfect,6 and
The title oligomers were prepared through the polymerization
chitosan7 have been used commercially and academically for gene between mono-substituted TACN and diglycidyl ether (see ESI† for
delivery. It should be noted, however, that despite remarkable details). Three diglycidyl ether bridges with varied rigidity (TOPh 4
development of cationic polymeric gene vectors, therapeutic TOBn 4 TOHex) were introduced for the purpose of elucidation of
applications of such biomaterials remain limited as a result of the structure–activity relationship (SAR). Furthermore, an amide-
their inadequate safety and transfection performance.8
containing analog (TNPh) was also prepared for comparison.
One most used strategy to enhance the transfection efficiency 1H NMR (400 MHz) spectra of the four novel compounds were
(TE) and biocompatibility of polycationic gene vectors is the grafting in full accordance with the expected structures. MALDI-TOF MS
of functional groups, such as PEG,9 hydrophobic groups,10 and
targeting ligands.11 These modifications may improve the stability,
cellular interaction and uptake, endosomal escape, and intracellular
unpacking of the polyplexes. Among these methods, hydrophobic
modification of cationic polymers has been established as an
effective and straightforward approach to improve membrane
a Key Laboratory of Green Chemistry and Technology (Ministry of Education),
College of Chemistry, Sichuan University, Chengdu 610064, PR China.
E-mail: xqyu@scu.edu.cn, ji_zhang@163.com; Fax: +86-28-85415886
b State Key Laboratory of Oral Diseases, Sichuan University, Chengdu 610041,
PR China. E-mail: zhouxd@scu.edu.cn
† Electronic supplementary information (ESI) available: Experimental section,
Fig. 1 Chemical structures of TACN-based oligomers.
supplemental figures and compound spectra. See DOI: 10.1039/c4cc01210k
6454 | Chem. Commun., 2014, 50, 6454--6457
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aromatic oligomers (except TOHex) gave distinctly smaller
polyplexes, suggesting that the rigid aromatic group plays an
important role in DNA condensation. We speculate that the aromatic
groups might strengthen the interactions between the molecules via
‘‘face-to-face’’ p–p stacking, which leads to particles with smaller
diameter.21 Zeta-potentials of the polyplexes increased along with
the increase of w/w ratio, and the surface charge was observed
about +15–25 mV above w/w of 6 (Fig. 3B). The size and zeta
potential for all oligomers became stable from w/w of B6,
indicating that full condensation was achieved.
Fig. 2 (A) Agarose gel electrophoresis of cationic oligomers complexed
with DNA to form polyplexes at different weight ratios. In each image, the
first lane is the DNA control. (B) Fluorescence quenching assay of EB/DNA
by addition of title oligomers.
The stability of polyplexes is essential for gene delivery.
A qualified vector should protect DNA from degradation caused
by nuclease, and resist the competitive binding of negatively
charged proteins. Agarose gel electrophoresis results (Fig. S1,
ESI†) revealed that in the presence of heparin, TOBn and
TOHex could no longer effectively condense DNA, and obvious
DNA release was observed. However, DNA retardation and protection
was retained for the polyplexes formed from TOPh and TNPh, and
no DNA degradation was observed in the presence of DNase. Such
difference should also be ascribed to the more rigid aromatic
structures of TOPh and TNPh, and their consequent stronger
interaction with DNA. By contrast, the more flexible PEI cannot
maintain DNA condensation in the presence of heparin.
The in vitro gene transfection efficiency of the new poly-
plexes was initially tested in HEK 293 cells by using pGL-3
plasmid as a luciferase reporter gene. Fig. 4A shows the relative
TE of these oligomers at various w/w ratios in comparison with
PEI 25 kDa at its optimal w/w ratio of 1.4. Accordingly, oligomer
with more rigid structure gave higher TE. At the optimal w/w
ratio, TOPh and TNPh exhibited 35.6 and 4.7Â higher TE than
PEI, respectively. This advantage might be attributed to their
higher DNA binding ability and the higher stability of the
relative polyplexes. On the other hand, TOBn, having only two
results revealed similar molecular weights (Mw E 4800) and low
polydispersity indexes (PDI: 1.03–1.11) for all oligomers.
The polyplexes formed at various oligomer/DNA weight
ratios (w/w) were subjected to agarose electrophoresis to detect
the amount required to inhibit the migration of plasmids. As
shown in Fig. 2A, it was found that all the materials can
effectively bind DNA and completely retard DNA migration at
a w/w ratio of 8. Increase of the number of carbon atoms in the
bridge (TOBn) might result in lower positive charge density,
leading to relatively weaker DNA binding ability. To quantitatively
study the effect of the backbone structure of oligomers on their
DNA affinity, ethidium bromide (EB) exclusion assay was carried
out (Fig. 2B). Results showed that all newly synthesized linear
oligomers could dramatically decrease the fluorescence intensity
caused by the intercalation of EB toward DNA base pairs. With the
increase of weight ratio, the relative fluorescence intensity became
approximately stable at w/w ratios of B4, indicating a nearly full
DNA condensation. The oligomers with more rigid backbone
(TOPh and TNPh) showed better DNA binding ability, and TOHex,
with a flexible long chain bridge, showed obviously lower fluores-
cence quenching ability. These findings are in agreement with
theoretical calculations on the simulations of the interactions
between rigid/flexible dendrimers and nucleic acids.20 Delicate
changes from ester to amide on the side chain of TACN may
slightly increase the DNA binding ability, which is in accordance
with the results obtained in gel retardation assays.
The properties of the formed polyplexes were first studied by
using dynamic light scattering (DLS) to measure their particle
sizes and zeta potentials. As shown in Fig. 3A, all four oligomers
are able to condense DNA into nanosized particles (B50 to
B400 nm) at w/w ratios ranging from 6 to 40. Interestingly,
Fig. 4 (A) Luciferase gene expression transfected by polyplexes at
different weight ratios in comparison with PEI (w/w = 1.4, N/P = 10) in
HEK 293 cells in the absence of serum. Data represent mean Æ SD (n = 3).
(B) Fluorescence microscope image of pEGFP-transfected HEK 293 cells in
the absence of serum at optimal weight ratio. (C) Transfection in HepG2
and HeLa cells.
Fig. 3 Particle size (left) and zeta-potential (right) of polyplexes obtained
at various weight ratios by DLS. Data represent mean Æ SD (n = 3).
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more methylene group in each unit, gave dramatically lower TE red fluorescence intensity observed through confocal laser
than its analog TOPh. For the most flexible compound TOHex, scanning microscopy (CLSM, Fig. 5A, B, F and G). In these cases,
poor TE was obtained. Green fluorescent protein (GFP) reporter the red pGL-3 plasmids mainly located around the blue-labelled
gene transfection gave similar results, which are shown in nuclei, which may facilitate nucleus importation and gene
Fig. 4B. The density of transfected cells by TOPh and TNPh expression. The images obtained from the transfection in
(especially TOPh) was much higher than that obtained in the serum-containing medium further confirmed the superiority of
experiment involving PEI. Subsequently, these two superior these two vector materials in gene delivery (Fig. S3, ESI†).
materials were applied to other cell lines including HepG2 Furthermore, flow cytometry assays were also performed to verify
and HeLa cells. As shown in Fig. 4C, it was interesting to find the higher cellular uptake in the TOPh/TNPh-involved transfection
that unlike the transfection toward HEK 293 cells, TNPh gave (Fig. S4, ESI†). In HEK 293 cells, the percentages of Cy5-positive
much higher TE than TOPh in tumor cells, especially in HepG2 cells were found to be 490%, which was much higher than that
cells, in which near 100 times higher TE than PEI was obtained. obtained by using PEI. Similar results were obtained in HepG2
Since different cell types possess varied transfection pathway cells, in which slightly lower uptake was found.
and cell apoptosis feature,22 the different TE of the title
The cytotoxicity of these polymers was estimated in three
oligomers between various cell lines may be acceptable. By tested cell lines by MTT assays (Fig. S5, ESI†). Results showed
comparing the transfection performance of TOPh and TNPh, it that these materials have much lower cytotoxicity than PEI,
could be confirmed that subtle structural change may have especially TOPh at high concentration. This might be due to
considerable influence on the TE, no matter what type of cell. their lower molecular weights. It is noteworthy that TNPh
The detailed transfection mechanisms leading to such differences showed distinctly higher cytotoxicity than TOPh, explaining
are under further investigation.
that the optimal transfection w/w ratio for TNPh is much lower
The gene transfection involving TOPh and TNPh in the than for TOPh.
presence of 10% of serum was also studied. Results showed
In summary, a series of linear oligomers with varied flexibility
that although the TE decreased in each case, both oligomers were synthesized. The oligomers with rigid aromatic backbone
still exhibited higher TE than PEI in all three cell lines (Fig. S2, were first applied to gene delivery and showed better DNA binding
ESI†). The good stability of the polyplexes formed from these two ability than the flexible analogues. In vitro transfection experiments
oligomers containing a rigid aromatic backbone may favour the also revealed that the materials with more rigid backbone (TOPh
resistance to serum-induced aggregation or premature DNA and TNPh) exhibited considerably higher TE, which were much
release.23 It is worth mentioning that in all tested cell lines, higher than ‘‘golden standard’’ 25 kDa branched PEI. Meanwhile,
TOPh gave higher TE than TNPh. Comparing the results these materials displayed lower cytotoxicity than PEI, showing
obtained in the transfection experiments without serum, it could better biocompatibility. It was also shown that subtle structural
be concluded that TOPh has higher serum tolerance than TNPh. diversity on the side chain of TACN may have a large influence
This might be attributed to its lower positive zeta-potential and on the TE and cytotoxicity. These materials containing aromatic
cytotoxicity (Fig. S5, ESI†).
backbones were proved to be promising gene carriers in the further
To obtain insight into the intracellular fate of the polyplexes, SAR study and in vivo application.
transfection experiments were performed using Cy5 labeled
This work was financially supported by the National Program on
DNA in HEK 293 and HepG2 cells (Fig. 5). As expected, TOPh Key Basic Research Project of China (973 Program, 2012CB720603),
and TNPh showed considerably higher efficiency to transport the National Science Foundation of China (No. 21232005,
pGL-3 plasmids into the cells than the flexible analogues and 51133004, J1310008 and J1103315), State Key Lab of Oral Diseases
PEI. This enhanced cellular uptake was revealed by the larger (Sichuan University) (SKLODOF2014OF01) and the Specialized
Research Fund for the Doctoral Program of Higher Education
(20120181130006) in China. J. Z. thanks the Program for New
Century Excellent Talents in University (NCET-11-0354).
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