Aza-crown ether locked on polyethyleneimine: Solving the contradiction between transfection efficiency and safety during: In vivo gene delivery

Article abstract of DOI:10.1039/c9cc10041e

We proposed a method using an aza-crown ether derivative to lock a hyperbranched polyethyleneimine, which endows the PEI_25k with tumor targeting ability, anti-serum ability and extended circulation in the blood meanwhile retaining the high gene complexation and high transfection efficiency. The method we proposed here simultaneously endows cationic materials with high transfection efficiency and high safety, which greatly pushed the cationic materials to be applied in in vivo gene delivery.

Full text of DOI:10.1039/c9cc10041e

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Aza-Crown Ether Locked on Polyethyleneimine: Solve the
Contradiction between Transfection Efficiency and Safety during
In Vivo Gene Delivery
Received 00th January 20xx,
Accepted 00th January 20xx
†
†
Shengran Lia, b, , Lin Linc, , Wenliang Wangd, Xinxin Yana, b, Binggang Chena, b, Sangni Jianga, b
Sanrong Liu*, a, Xiaojing Ma*, a, Huayu Tianb, c, Xifei Yu*, a, b
,
DOI: 10.1039/x0xx00000x
www.rsc.org/
We proposed a method using an aza-crown ether derivative to lock delivery vectors with both high transfection efficiency to cancer
on hyperbranched polyethyleneimine, which conferring PEI25k cells and low toxicity to healthy cells.6
tumor targeting, anti-serum and extended circulation in the blood,
meanwhile retaining the high gene complexation, high transfection
efficiency of PEI25k. The method we proposed here endows cationic
materials with high transfection efficiency and high safety
simultaneously, which greatly pushes the cationic materials to be
applied in vivo gene delivery.
Researchers developing gene delivery vectors face a substantial
challenge: vectors with high transfection efficiency exhibit high
cytotoxicity.1 Cationic materials, specifically hyperbranched
polyethyleneimine (PEI25k), have been investigated as gene
carriers because of their superior gene complexing ability and
high transfection efficiency.2 However, these cationic materials
exhibit high cytotoxicity due to the lack of tumor targeting,
serum resistance, and extended circulation in the blood.3 This
toxicity severely limits their further in vivo application. Currently,
polyethylene glycol (PEG) is grafted onto cationic materials to
shield their binding to cell membranes, thus allowing the
complex to accumulate at tumor sites.3a, 4 Although the increase
of grafted PEG leads to longer blood circulation, it reduces the
gene complexing ability and transfection efficiency of the
cationic materials because of the reduction of the positive
charge density.5 Therefore, it is essential to determine a method
to overcome these current limitations and generate gene
Scheme 1. Schematic of how the ACE-GE11 locks on PEI25k
to shield endocytosis in the blood circulation and unlocks at
a. Laboratory of Polymer Composites Engineering, Changchun Institute of Applied
Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China. E-mail:
liusanrong@ciac.ac.cn, xjma@ciac.ac.cn, xfyu@ciac.ac.cn.
tumor sites.
Crown ethers, cyclic organic compounds composed of several
ether units, are capable of binding cations7 and have been
explored as phase transfer catalysts, for metal extraction and
b. University of Science and Technology of China, Hefei, Anhui 230026 China.
c. Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry,
Chinese Academy of Sciences, Changchun, Jilin 130022, China.
d. Department of Neurology, Johns Hopkins University School of Medicine,
Baltimore, MD 21205, USA
9
supramolecular chemistry,8 and as fluorescence probes.7,
† The authors contributed equally to this work
These properties inspired us to combine crown ethers with
abundant amino cations in cationic materials to prevent the
binding of cationic materials to cell membranes via steric
hindrance. We altered the structure of the crown ether to aza-
Electronic Supplementary Information (ESI) available: [Experimental details,
characterization, and et al.]. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
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18-crown-6 ether (ACE) that can be detached from the cationic
materials when exposed in the acidic environment of the tumor
sites. Moreover, we linked GE11 (Y-H-W-Y-G-Y-T-P-Q-N-V-I), an
EGFR over-expressed cancer cell targeting peptide,10 to the ACE
to make it an active target. We chose PEI25k as a primary
transfection reagent due to its well-known gene complexing
ability and transfection efficiency. As shown in Scheme 1, the
previously prepared ACE-GE11 was mixed with the PEI25k/DNA
complex to obtain the final nanoparticle. The working principle
of this nanoparticle is as follows: the crown ether in ACE-GE11
can be stably combined with the outermost amino cation of the
PEI25k/DNA complex by host-guest interaction and hydrogen
bonds.11 The amino cations locked by ACE-GE11 will neither
bind to the cell membrane nor bind to proteins in the blood due
to the steric hindrance surrounding the amino cations. This
facilitates the nanoparticles of ACE-GE11/(PEI/DNA) to be
stable and remain circulating in the blood for an extended
period. The targeting peptide GE11 could lead the nanoparticles
to accumulate at the tumor site. When the nanoparticles arrive
at the tumor site, the tertiary amines on the ACEs would
partially become positively charged due to the slightly acidic
environment of the tumor sites. Thereafter, the ACE-GE11
would detach from PEI/DNA complex owing to charge repulsion
with the amino cations on PEI25k, making the nanoparticles
more susceptible to endocytosis by cancer cells. After
endocytosis, the tertiary amines on the ACEs would be
completely protonated and detach from PEI/DNA complex
while entering the lysosome. The nanoparticles of PEI/DNA
were entirely exposed and showed high transfection efficiency
and toxicity to kill the cancer cells. Compared with the widely
used PEG or other methods, the nanoparticle prepared using
the ACE derivative is smaller sized with more function
(shielding, targeting, and detaching). It is more convenient to
prepare (physically mixed ACE with PEI/DNA rather than
chemical modification12) and has better shielding effect (one
ACE shields one amino cation). Finally, it detaches faster in
tumor sites by electrostatic repulsion rather than chemical
bond rupture.13 These properties together are more practical
during gene delivery.
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DOI: 10.1039/C9CC10041E
Fig. 1. (a) Size of the nanoparticles of ACE-GE11/(PEI/DNA)
detected by DLS and TEM; scale bar: 100 nm. (b) The binding
kinetics of ACE on PEI. Lock ratio is the molar ratio of ACE /
+
(-NH3 on PEI). (c) The ratio of ACE unlocked from PEI25k at
different pH in 2 h. (d) GFP gene transfection of the
complexes of our materials and DNA at N/P=10 on NIH-3T3
and A549 cells. Luciferase gene transfection of the
complexes of our materials and DNA at different N/P ratios
on (e) A549 cells and (f) NIH-3T3 cells.
ACE was eluted by CH2Cl2 to calculate the binding kinetics (Fig.
1b). This calculation was based on the ratio of primary,
secondary, and tertiary amines on PEI25k, which was 1:2:1. Only
primary amines protonated in water could be combined with
aza-18-crown-6 ether (ACE) by host-guest interaction and
hydrogen bonds, while other amino cations on PEI could
not.11,15 We found that 83% of -NH3+ on PEI was locked by ACE
within 30 minutes, and the limit of the lock ratio was 86%. We
also used NMR to monitor the binding process, and found the
lock ratio could be 82% in 30 minutes (Fig. S11). We detected
the binding affinity of ACE on PEI in NaCl solutions (Fig. S12),
and we found the lock ratio remained 83% in 1% NaCl solution
(NaCl content in blood is 0.9%). This strong binding affinity was
due to the formation of hydrogen bonds between ACE and PEI
reference. We added ACE/PEI complex to water with different
pH, dialyzed to remove the free ACE, then analyzed by NMR (Fig.
S13). The results are shown in Fig. 1c. We found that 20% of ACE
detached from PEI25k at pH=6.0-6.5 and 100% at pH=5.0-5.5 in 2
h due to the protonation of tertiary amine on ACE in an acidic
environment and electrostatically repulsion from the amino
cations on PEI25k. This result indicated that when our
nanoparticles of ACE-GE11/PEI/DNA arrive at the tumor area
(pH=6.0-6.5), ACE-GE11 would partially detach from the
nanoparticles to enhance the endocytosis of the nanoparticles.
After entering the lysosome, the ACE-GE11 would completely
detach from the nanoparticles, thus maximizing the
The product ACE-GE11 was synthesized according to the
procedure described in Scheme S1 (see the supporting
information) and was characterized by NMR (Fig. S1-S6) and
MALDI-TOF-MS (Fig. S7) analysis. The complex of PEI25k/DNA
showed optimal size (70±2 nm) and zeta potential (+23±3 mV)
at N/P=10 compared with other N/Ps,14 which was detected by
dynamic light scattering (DLS) (Fig. S8, S9). After mixing ACE-
GE11 with PEI25k/DNA, the size of the nanoparticles of ACE-
GE11/(PEI/DNA) was 60 nm (Fig. 1a), which was detected by
transmission electron microscope (TEM), while the zeta
potential of the nanoparticles decreased to +17 mV (Fig. S9).
The changes of size and charge indicated ACE-GE11 was bound
to PEI/DNA successfully.
To investigate the lock and unlock ability of ACE-GE11 on
amino cations of PEI25k, we used ACE to mix with PEI25k. The
ACE/PEI complex was dialyzed with phosphate buffered saline
transfection efficiency of PEI25k
. We also explored the
detachment speed of ACE from PEI under acidic conditions and
found that 44% of ACE were detached at 30 min and were
almost fully detached in 1 h (Fig. S14, S15). This acidic-response
speed was much faster than chemical bond cleavages, such as
acetal bonds and hydrazone bonds.16
(PBS) to remove the ACEs that were not combined to PEI25k
.
After dialysis, the complexes of ACE/PEI were freeze-dried and
2 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C9CC10041E
Fig. 2. (a) CLSM images of NIH-3T3 and A549 cells cultured
with ACE-GE11/(PEI25k/cy5-DNA) complexes for 1 h; cy5-
DNA and cell nuclei stained with DAPI are shown in red and
blue, respectively; scale bars: 20 μm. GFP-positive A549
cells (b) and NIH-3T3 cells (c) measured by flow cytometry
after treatment with the complexes of our materials and
DNA with or without serum. (d) Luciferase gene
transfection of the complexes of our materials and DNA at
N/P=10 with different amount of FBS. Cell viability of (e)
NIH-3T3 cells and (f) A549 cells incubated with varying
concentrations of PEI25k and ACE-GE11/PEI25k for 24 h; the
Fig. 3. (a) In vivo fluorescence imaging of A549 tumor
bearing mice at different times after intravenous injection
of the complexes of our materials and cy5-DNA; Ex vivo
fluorescence imaging of various organs and tumors excised
from the mice after injection for 24 h (the right). (b) The
pharmacokinetics of cy5-DNA in the blood after the mice
were intravenously injected with different samples. (c)
Tumor growth curves in A549 tumor bearing mice treated
with different samples; DNA: caspase-3 1 mg/kg; PEI/DNA:
N/P=10; the sample ACE-GE11/PEI: PEI 3 mg/kg. (d)
Representative images of the tumors via different
treatments after 18 days. (e) H&E analysis of the tumors
after 18 days of treatment via different reagents. Scale
bars: 50 μm.
abscissa refers to the concentration of PEI25k
.
The effects of ACE-GE11 on shielding healthy cells
transfection and targeting cancer cells were measured using
green fluorescence protein reporter gene (pGFP). We found
PEI25k showed high transfection efficiency on mouse embryo
fibroblast (NIH-3T3) cells and human lung cancer (A549) cells
(
Fig. 1d). After PEI25k was locked by ACE or ACE-GE11, the
transfection efficiency of them decreased to quite low in
healthy (NIH-3T3) cells, while increased much in cancer (A549)
cells. We found the nanoparticles of ACE-GE11/(PEI/DNA) were
shielded and could not be endocytosed by NIH-3T3 cells
regardless of the N/P ratio (Fig. S16). These nanoparticles could
be endocytosed by A549 cells and showed higher transfection
efficiency than the nanoparticles of PEI/DNA (Fig. S17). Then, a was higher than PEI/DNA on A549 cells (Fig. 2b), while the ACE-
luciferase plasmid (pGL3) was used as a reporter gene to GE11/(PEI/DNA) showed decreased luciferase expression in
evaluate the transfection efficiency of our materials. We found NIH-3T3 cells (Fig. 2c). The GFP, luci-expression, CLSM, and flow
that the nanoparticles of ACE-GE11/(PEI/DNA) showed a higher cytometry results were consistent, which indicated the ACE-
transfection efficiency than PEI/DNA at all N/P ratios in A549 GE11 could inhibit healthy cell endocytosis of PEI/DNA while
cells
GE11/(PEI/DNA) decreased at least 60 times compared with
PEI/DNA in NIH-3T3 cells due to the shield effect of ACE-GE11 A549 cells to investigate the ability of serum resistance of our
Fig. 1f). nanoparticles. ACE-GE11/(PEI/DNA) showed high luciferase
We used fluorescence labeling of DNA (cy5-DNA) and expression with 10%, 30%, and 50% FBS, while PEI/DNA showed
(
Fig. 1e). The transfection efficiency of ACE- targeting cancer cells.
We added 10%, 30%, and 50% fetal bovine serum (FBS) to
(
confocal laser scanning microscope (CLSM) to trace the few luci-expression with FBS (Fig. 2d). The GFP results shown in
transportation of genes by our nanoparticles. The results Fig. S21 indicated PEI/DNA locked by ACE-GE11 could resist
showed that the nanoparticles of ACE-GE11/(PEI/DNA) were medium with 30% FBS, while PEI/DNA could not resist any
quickly endocytosed and delivered to the nucleus in 1 h by A549 amount of FBS.
cells, while they were not endocytosed by NIH-3T3 cells (Fig. 2a).
To verify the ACE-GE11 could substantially improve the safety of
The detailed time-dependent fixed CLSM studies of gene PEI25k, we evaluated the cytotoxicity of our materials by Celltiter-Blue
transportation by ACE-GE11/(PEI/DNA) on NIH-3T3 cells and assay. ACE-GE11 showed no toxicity to NIH-3T3 cells, even under the
A549 cells are shown in Fig. S18-S20. The flow cytometry results concentration of 200 μg/mL (Fig. S22). PEI25k had potent cytotoxicity
showed that the luciferase expression of ACE-GE11/(PEI/DNA) to NIH-3T3 cells and A549 cells, and cells could not survive at 10
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μg/mL (Fig. 2e, f). However, after PEI25k locked by ACE-GE11, 100% the tumor sites. The method we proposed breaches the barrier
View Article Online
DOI: 10.1039/C9CC10041E
of NIH-3T3 cells survive at the concentration of 40 μg/mL of PEI25k between transfection efficiency and safety in gene carriers and
(Fig. 2e). Interestingly, ACE-GE11/PEI25k showed no A549 cancer cells shows promise for in vivo gene therapy.
survive at 40μg/mL of PEI25k (Fig. 2f). The results indicated that ACE-
GE11 could significantly improve the safety of PEI25k to healthy cells
while maintaining substantial toxicity to cancer cells. The hemolysis
Methods and Experimental
test confirmed ACE-GE11/(PEI/DNA) showed no cytotoxicity to red
blood cells even at 150 μg/mL while PEI/DNA killed red blood cells at
40 μg/mL (Fig. S23, S24).
The animal experiments were approved by the Animal Care and
Use Committee of Northeast Normal University. All operations
were performed according to international guidelines
concerning the care and treatment of experimental animals.
The case number of institutional approval of the animal is
SCXK(Jing)2016-0006.
The tumor accumulation of our materials was studied by tail-
vein injection into an A549 xenograft mouse model, and the
fluorescence images were recorded at different times (Fig. 3a).
The nanoparticles of ACE-GE11/(PEI/DNA) showed substantial
accumulation at 12 h after injection and continued to 24 h. The
nanoparticles of PEI/DNA did not accumulate at tumor sites.
The tumor and main organs of the two groups were harvested
at 24 h post-injection (Fig. 3a). Most of the nanoparticles of
PEI/DNA were accumulated at the liver site, while most of the
ACE-GE11/(PEI/DNA) accumulated at the tumor site and
showed little accumulation in the liver. Pharmacokinetic
analysis determined ACE-GE11/(PEI/DNA) owned longer
circulation time in blood than PEI/DNA (Fig. 3b).
To investigate the antitumor efficacy of our materials, the
A549 tumor-bearing mice were administered different samples
intravenously. We found the tumors growth of mice injected
with ACE-GE11/(PEI/DNA) were efficiently suppressed, and the
tumor size diminished. The mice treated with PEI/DNA had
tumors that continued to increase (Fig. 3c, d and S25). The
Conflicts of interest
There are no conflicts to declare.
Notes and references
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tumor suppression ratio was calculated and shown in Fig. S26
,
and ACE-GE11/(PEI/DNA) showed high tumor suppression to
97.8%.17 We also treated the mice with ACE-GE11/PEI at the
dose of PEI25k was 3 mg/kg without DNA. Interestingly, we found
the tumor growth was efficiently suppressed (Fig. 3c, d and S25),
and the mice remained healthy. This result suggested that the
PEI25k locked by ACE-GE11 was not toxic to healthy cells, but
highly toxic to cancer cells at a large dose of PEI25k. The mice
treated with ACE-GE11 locked materials were healthy and their
body weights were maintained at 22-23 g, whereas the weight
of the mice treated with PBS or PEI/DNA significantly decreased
5
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Fig. S27). Hematoxylin and eosin (H&E) assays were used to
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Fig. 3e, the ACE-GE11 locked materials exhibited marked
necrosis of tumor cells, and negligible damage to healthy organs
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Conclusions
In conclusion, we tested a method that used aza-crown ether
derivatives to lock on PEI25k to confer it tumor targeting, anti-
serum and extended blood circulation, meanwhile maintain
transfection efficiency and gene complexing ability of PEI25k
.
Compared with the widely used PEG-modified or other
methods, the ACE derivative was smaller with more
functionality and convenient usage by mixing it with cationic
materials. Furthermore, ACE showed better shield effect in the
blood and faster detachment from the complex when reaching
16 F. Seidi, R. Jenjob, D. Crespy, Chem. Rev. 2018, 118, 3965-
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17 H. Fang, J. Chen, L. Lin, F. Liu, H. Tian, X. Chen, ACS Appl.
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