pH-Sensitive Polycations for siRNA Delivery: Effect of Asymmetric Structures of Tertiary Amine Groups

Article abstract of DOI:10.1002/mabi.202100025

pH-sensitive polyelectrolytes provide enormous opportunity for siRNA delivery. Especially, their tertiary amine structures can not only bind genes but also act as pH-sensitive hydrophobic structure to control genes release. However, the influence of molecular structures on siRNA delivery still remains elusive, especially for the asymmetric alkyl substituents of the tertiary amine groups. Herein, a library of N-methyl-N-alkyl aminoethyl methacrylate monomers (MsAM) with asymmetric alkyl substituents on the tertiary amine group is synthesized and used to prepare a series of tri-block polycationic copolymers poly(aminoethyl methacrylate)-block-poly (N-methyl-N-alkyl aminoethyl methacrylate)-block-poly(ethylene glycol methacrylate) (PAMA-PMsMA-PEG). And the properties of these polycations and their self-assembled micelles are characterized, including molecular structure, proton buffering capacity, pH-sensitivity, size, and zeta potential. With the length increase of one alkyl substituent, the proton buffering capacity of both monomers and polycations is demonstrated to be narrowed down. The siRNA delivery efficiency and cytotoxicity of these micelles are also evaluated on HepG2 cells. In particular, poly(aminoethyl methacrylate)-block-poly(N-methyl-N-ethyl aminoethyl methacrylate)-block-poly(ethylene glycol methacrylate) (PAMA-PMEMA-PEG) elicited the best luciferase knockdown efficiency and low cytotoxicity. Besides, PAMA-PMEMA-PEG/siRRM2 also induced significant anti-tumor activity in vitro. These results indicated PAMA-PMEMA-PEG has potential for further use in the design of gene vehicles with the improved efficiency of siRNA delivery.

Full text of DOI:10.1002/mabi.202100025

ReseaRch aRticle
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pH-Sensitive Polycations for siRNA Delivery: Effect of
Asymmetric Structures of Tertiary Amine Groups
Qinping Yang, Yanliang Dong, Xuanyu Wang, Zhihao Lin, Mingyu Yan, Weiwei Wang,
Anjie Dong, Jianhua Zhang,* Pingsheng Huang,* and Changrong Wang*
1
. Introduction
pH-sensitive polyelectrolytes provide enormous opportunity for siRNA
delivery. Especially, their tertiary amine structures can not only bind genes but
also act as pH-sensitive hydrophobic structure to control genes release. How-
ever, the influence of molecular structures on siRNA delivery still remains
elusive, especially for the asymmetric alkyl substituents of the tertiary amine
groups. Herein, a library of N-methyl-N-alkyl aminoethyl methacrylate mono-
mers (MsAM) with asymmetric alkyl substituents on the tertiary amine group
is synthesized and used to prepare a series of tri-block polycationic copoly-
mers poly(aminoethyl methacrylate)-block-poly (N-methyl-N-alkyl aminoethyl
methacrylate)-block-poly(ethylene glycol methacrylate) (PAMA-PMsMA-PEG).
And the properties of these polycations and their self-assembled micelles
are characterized, including molecular structure, proton buffering capacity,
pH-sensitivity, size, and zeta potential. With the length increase of one alkyl
substituent, the proton buffering capacity of both monomers and polycations
is demonstrated to be narrowed down. The siRNA delivery efficiency and
cytotoxicity of these micelles are also evaluated on HepG2 cells. In particular,
poly(aminoethyl methacrylate)-block-poly(N-methyl-N-ethyl aminoethyl
methacrylate)-block-poly(ethylene glycol methacrylate) (PAMA-PMEMA-PEG)
elicited the best luciferase knockdown efficiency and low cytotoxicity. Besides,
PAMA-PMEMA-PEG/siRRM2 also induced significant anti-tumor activity in
vitro. These results indicated PAMA-PMEMA-PEG has potential for further
use in the design of gene vehicles with the improved efficiency of siRNA
delivery.
Small interfering RNA (siRNA), non-
coding RNAs of 20–25 bp in length,
can specifically suppress expression of
target genes, which have shown great
potentials for a great many diseases.
[
1]
[2,3]
Recently, several nuclei acid drugs have
[
4]
been applied to clinical trials. However,
the degradability by nuclei acid enzyme
and electronegativity had serious impacts
on siRNA systematic and intracellular
delivery.
[5,6]
Therefore, developing an effi-
cient siRNA delivery vehicle is extremely
urgent.
Traditionally, the siRNA vehicles can
be divided into two categories: virus vec-
tors and non-virus vectors. Although viral
vectors own high transfect efficiency, they
also exhibit significant limitations, such
as immunogenicity, limited load capacity,
confined cytotropism, and complex ana-
lytical and production processes.
importantly, the safety of viral vectors still
limits their clinical trials.
[
7,8]
More
[
9,10]
Non-viral
vehicles, as the viral vehicles succeda-
neum, have the advantages of low immu-
nogenicity, low toxicity, and low cost.
[
11–13]
Among the non-viral vehicles, the polyca-
tions have become the outshining tactics
for gene delivery due to their controlled
Q. P. Yang, Y. L. Dong, X. Y. Wang, Z. H. Lin, M. Y. Yan, Prof. A. J. Dong,
Prof. J. H. Zhang
Department of Polymer Science and Engineering
School of Chemical Engineering and Technology
Tianjin University
Tianjin 300072, China
E-mail: jhuazhang@tju.edu.cn
Dr. C. R. Wang
School of Pharmacy
Shandong New Drug Loading & Release Technology and Preparation
Engineering Laboratory
Binzhou Medical University
Yantai 264003, China
E-mail: WCR20142014@163.com
Prof. W. W. Wang, Dr. P. S. Huang
Tianjin Key Laboratory of Biomaterial Research
Institute of Biomedical Engineering
Chinese Academy of Medical Sciences and Peking Union Medical
College
Tianjin 300192, China
E-mail: sheng1989.2008@163.com
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/mabi.202100025.
DOI: 10.1002/mabi.202100025
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preparation and flexible modification,[14] such as poly(2-dimeth-
Meanwhile, strategies such as stability, surface, and size
transitions (3S Transitions) were proposed to resolve those
dilemmas, which is to create efficient and low-toxicity nonviral
gene vectors. However, The lower efficiency of cytosolic delivery
[15,16]
ylaminoethyl methacrylate) (PDMAEMA),
polyethylen-
imine (PEI),[ poly(L-lysine) (PLL),
17]
[18,19]
and polyamidoamine
dendrimer (PAMAM),[ which could condense gene through
charge interaction to form nanosized polyion complex (PIC).
Such kind of vectors with abundant amine had presented excel-
lent siRNA binding capacity and delivery efficiency, but the
additional charge caused low biocompatibility and high bio-
toxicity. To evade the cytotoxicity induced by surplus charge,
20]
[
35,36]
of siRNA still is the bottleneck.
In some previous studies, a library of polycationic micelles
consisted of various hydrophobic sections with different pKa
[
37]
were evaluated on siRNA delivery,
and a significant dis-
covery was that the pKa values of hydrophobic segments in
the range of 5.8–6.2 induced higher siRNA delivery efficiency
PEGylated strategies were generally brought to embellish poly-
cations.[
21,22]
PEGylation polycations could improve biocompat-
in vitro and in vivo. These results indicated that the tertiary
[38]
[23]
ibility, stability and prolong the circulation. However, several
shortcomings were introduced by PEGylation, named “PEG
dilemma”, which not only hampered the interaction between
siRNA and polycations but also inhibited the cell uptake and
escape abilities.[ In order to overcome the drawback of PEG
dilemma, many approaches were adopted, such as integrating
amine structures can not only bind genes but also act as pH-
sensitive hydrophobic structure to exert significant influence
on the siRNA delivery. However, the influence of molecular
structures of the tertiary amine groups still remains remark-
ably elusive, especially for the asymmetric alkyl substituents.
Herein, a new series of N-methyl-N-alkyl aminoethyl meth-
acrylate monomers (MsMA, Alkyl = methyl, s = M; Alkyl =
ethyl, s = E; Alkyl = propyl, s = P; Alkyl = butyl, s = B; and
Alkyl = amyl, s = A), with asymmetric alkyl substituents on
the tertiary amine groups, were synthesized and shown in
Scheme 1. And the pH sensitivities of these monomers and
the corresponding polymers were studied. The results reflected
that the proton buffering capacity was narrowed down with
the length increase of one alkyl chain. Furthermore, five
tri-block polycations, poly(aminoethyl methacrylate)-block-
poly(N-methyl-N-alkyl aminoethyl methacrylate)-block-poly
(ethylene glycol methacrylate) (PAMA-PMsMA-PEG) were syn-
thesized by reversible addition-fragmentation chain transfer
(RAFT) polymerization. The physicochemical properties and
24]
[24,25]
sheddable PEG
and introducing hydrophobization sec-
tions.[ More importantly, the hydrophobization of polycations
26]
could not only resolve the PEG dilemma but also stabilize the
PIC.[
21,22,27]
Particularly, the introduction of pH-sensitive sec-
tions into polycations to form pH-sensitive polycationic vehi-
[28]
cles has drawn much attention, recently.
The pH-sensitive
polycations are in favor of siRNA delivery, which is explained by
29,30]
“
proton sponge effect”.[
The amine groups of pH-sensitive
section in polycations were protonated in acid endosome and
induced the changes of osmotic pressure, resulting in the dis-
ruption of endosomal membrane and thus release of the cargo
into cytosol.[ Shen et al.
31]
[32–34]
had analyzed the cancer gene-
delivery cascade and the barriers, the needed nanoproperties.
Scheme 1. Scheme diagrams of A) synthesis routes of pH-sensitive monomers with asymmetric N-alkyls substituents and B) formation of PAMA-
PMsMA-PEG/siRNA micelleplexes with pH-responsive release of siRNA.
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1
Figure 1. The H NMR spectra of MsMA monomers in CDCl .
3
bio-experiments of these polycations as siRNA vehicles in
vitro were investigated. Compared with other polycations,
N-methyl-N-alkyl ethanolamine and methacryloyl chloride. The
molecular structures of the obtained MsMA were characterized
1
1
poly(aminoethyl
methacrylate)-block-poly(N-methyl-N-ethyl
by H NMR, as shown in Figure 1. All H NMR spectra showed
the characteristic peaks of carbon-carbon double bond at about
5.6 and 6.1 ppm and the methyl group on double bond at about
1.9 ppm. In addition, the characteristic peaks of N-alkyls sub-
stituents on all MsMA monomers can be clearly observed.
These results indicated that all of the MsMA monomers with
high purity were successfully synthesized. Furthermore, the
reaction yield of MEMA, MPMA, MBMA, and MAMA were
aminoethyl methacrylate)-block-poly (ethylene glycol meth-
acrylate) (named as PAMA-PMEMA-PEG) with pKa values of
6
.2 was found to possess higher gene knockdown efficiency,
better biocompatibility, and higher anti-tumor efficiency in
vitro, which can be used as an efficient siRNA delivery vehicles.
8
3%, 77%, 76%, and 80%, respectively.
2
. Results and Discussion
2
.1. Synthesis and Characterizations of MsMA Monomers
2
.2. Synthesis and Characterizations of Tri-Block Polycations
A
series of polymerizable and pH-sensitive monomers,
PAMA-PMsMA-PEG
N-methyl-N-alkyl aminoethyl methacrylate with asymmetric
N-alkyls substituents, including N-methyl-N-ethyl aminoethyl
methacrylate (MEAM), N-methyl-N-propyl aminoethyl meth-
acrylate (MPAM), N-methyl-N-butyl aminoethyl methacrylate
Due to their versatile polyelectrolyte properties and pH-
responsive functions, the MsMA and its polymers are
expected to serve as promising nanocarriers for gene delivery.
In order to investigate the influence of the asymmetric alkyl
substituents of the tertiary amine groups on siRNA delivery,
five tri-block PAMA-PMsMA-PEG polycations were syn-
thesized by multi-step sequential RAFT polymerization, as
shown in Figure 2A. Firstly, the amino-protected P(N-(tert-
butoxycarbonyl) aminoethyl methacrylate) (PBAMA) with
pre-defined polymerization degree of about 50 was prepared
(
(
MBAM), and N-methyl-N-amyl aminoethyl methacrylate
MAAM) were designed and synthesized by nucleophilic sub-
stitution and esterification reaction, as shown in Scheme 1A.
Typically, N-methyl-N-alkyl ethanolamine was firstly synthe-
sized by the nucleophilic substitution between N-methyl etha-
nolamine and corresponding 1-bromo alkane. And then the
MsMA monomers were obtained by the reaction between
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1
Figure 2. A) Synthesis routes of PAMA-PMsMA-PEG and H NMR of PAMA-PMsMA-PEG in d-DMSO (B: PAMA-PMMMA-PEG, C: PAMA-PMEMA-PEG,
D: PAMA-PMPMA-PEG, E: PAMA-PMBMA-PEG, F: PAMA-PMAMA-PEG)
1
in the presence of S-Dodecyl-S′-(α,α′-dimethyl-α″-acetic
acid)-trithiocarbonate as chain transfer agent and AIBN as
initiator in DMF. The PBAMA with polymerization degree
of about 44 was isolated and purified by dialysis, and then
was used as a macro-RAFT agent in the subsequent polym-
erization. And then the MsMA as pH-sensitive hydrophobic
segment and polyethylene glycol methacrylate (EG) as hydro-
philic segment were selected as second and third monomer,
respectively, to be polymerized under similar condition to
prepare the tri-block copolymers. The polymerization degree
of each block was controlled by the initial molar mass ratio
of monomer to RAFT agent and monomer conversion. After
amino deprotection by TFA, the polycations (PAMA-PMsMA-
PEG) were obtained. To confirm the chain compositions and
the obtained polycations. As shown in Figure 2, all HNMR
spectra of PAMA-PMsMA-PEG show the characteristic peak
(f) corresponding to the PAMA segment at about 3.2 ppm,
characteristic peaks (g, h, and j) corresponding to the PMsMA
segment at about 2.7 and 1.5 ppm as well as characteristic
peak (i) corresponding to the PEG segment at about 3.5 ppm.
All characteristic signals of PAMA, PMsMA, and PEG units
1
could be seen clearly in the HNMR spectra, indicating the
successful preparation of PAMA-PMsMA-PEG. In addition,
all PAMA-PMsMA-PEG copolymers possess a relatively low
polydispersity (PDI ≈ 1.5). The polymerization degree cal-
1
culated from the ratio of H NMR peak area and molecular
weights obtained by GPC were listed in Table 1. The results
indicated that the triblock PAMA-PMsMA-PEG polymers
with controlled chain architecture and segment composition
1
molecular structures, HNMR and GPC were used to analyze
Table 1. Characterizations of PAMA-PMsMA-PEG.
AMAa
44
MsMAa)
EGa)
5.3
5.2
5.2
5.4
5.1
5
b)
PDIb)
1.57
1.47
Samples
Mn (×10 Da)
PAMA-PMMMA-PEG
PAMA-PMEMA-PEG
PAMA-PMPMA-PEG
PAMA-PMBMA-PEG
PAMA-PMAMA-PEG
88
87
82
90
97
2.56
2.53
2.23
2.68
2.78
44
44
1.50
1.49
1.53
44
44
a)
Polymerization degree calculated from H NMR; b)Determined by GPC.
1
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Figure 3. Acid-titration curves of MsMA monomers (A: MMMA, B: MEMA, C: MPMA, D: MBMA, E: MAMA) and F) consumption of NaOH solution,
−
1
MsMA concentration: 2 mg mL , NaOH concentration: 0.1 M.
as well as predetermined molecular weight were successfully
prepared by RAFT polymerization.
efficient endosomal disruption and facilitating the escape
[
37,39]
from endosomes.
The new class of MsMA and PAMA-
PMsMA-PEG with tunable buffering capacities were studied
by acid–base titration, which was widely used to determine the
[
38]
2
.3. Proton Buffering Capacity of MsMA and
buffering capacities of cationic materials and nanostructures.
PAMA-PMsMA-PEG
The samples were dissolved in HCl solution and titrated by
gradual addition of NaOH solution. The titration studies were
conducted over a pH range of 2–12 and shown in Figure 3A–E.
The acid-base titration results showed that, with the increase of
the length of the substituted alkyl chain, the proton buffering
As well known, the tertiary amine-containing compounds
and polymers with pKa of about 6 can undergo protonation
in the acidic endosomal environment, thus resulting in an
Figure 4. Acid-titration curves of PAMA-PMsMA-PEG polycations (A: PAMA-PMMMA-PEG, B: PAMA-PMEMA-PEG, C: PAMA-PMPMA-PEG, D: PAMA-
PMBMA-PEG, E: PAMA-PMAMA-PEG) and F) the pKa values of PAMA-PMsMA-PEG polycations.
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capacity of MsMA monomers occurred in a relatively nar-
rower pH range. The MsMA monomers were found to require
decreasing amounts of NaOH (Figure 3F) with increasing the
length of the substituted alkyl chain in MsMA (carbon number
from 1 to 5). It was possibly due to that longer alkyl chain could
improve the steric-hindrance effect, leading to the strengthened
hydrophobic interaction.
effect.[40] To assess the pH-sensitive ability of the series poly-
cations, the acid–base titration was further carried out and
shown in Figure 4. The concentration of polycations was 2 mg
−
1
mL . Based on the results in Figure 4A–E, the proton buff-
ering capacity also mainly occurred in a narrow pH range with
the same trend of MsMA monomers. The buffer capability
of PAMA-PMsMA-PEG could be attributed to the presence
of amine groups of MsMA. The buffer capability of cationic
PAMA-PMsMA-PEG polymers as gene carriers has been proved
to be favorable for the intracellular release and gene transfec-
tion. Meanwhile, it can be found from Figure 4F that the pKa
values declined from 6.9 to 5.2 with increase in the length of
the substituted alkyl chain, which was caused by the enhance-
ment of interhydrophobic force of pH-sensitive section. It was
worth noting that the PAMA-PMEMA-PEG possessed a pKa
The pKa values showed important influence on siRNA
delivery efficiency.[
37,39]
It has been reported that the pKa values
of amino lipids located in 6.2–6.5 elicited efficient delivery on
therapeutic agent siRNA.[ In our previous work, we found
that efficient gene knockdown was achieved by pH-sensitive
polycations with pKa values of hydrophobic sections at 5.8–6.2
in vitro and in vivo.[ Besides, we verified that pKa values
affected siRNA delivery efficiency through proton sponge
39]
37]
Figure 5. A) Sizes and B) zeta potentials and C) TEM images of PAMA-PMsMA-PEG micelles (scale bar: 200 nm). D) Gel electrophoresis analysis to
determine the siRNA binding capacities of PAMA-PMsMA-PEG micelles. (siRNA: 50 nM; siNC = Negative controlled siRNA).
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of 6.2, which meant that it might be very suitable for siRNA
delivery. Some works reported that the pH of endosome was
about 5.5–6.5.[ Therefore, the pKa values of 6.2 indicated
that PAMA-PMEMA-PEG micelles could be protonated in
endosome, thus resulting in the change of osmotic pressure,
which would facilitate the endosomal release of gene and thus
improve the transfection efficiency of siRNA.
rowed down proton buffering capacity of pH-sensitive sections
caused the less protonation of pH-sensitive segments, further
resulted in the lower zeta potentials under neutral conditions.
Moreover, the considerable zeta potentials results illustrated the
cationic micelles had the potential abilities of binding siRNA.
The electronegative siRNA could be bound by electroposi-
tive polycations through the charge interaction. To confirm the
siRNA binding abilities of PAMA-PMsMA-PEG micelles, the
gel electrophoresis analysis was implemented. As shown in
Figure 5D, PAMA-PMMMA-PEG, PAMA-PMEMA-PEG, PAMA-
PMPMA-PEG, and PAMA-PMBMA-PEG micelles could bind
siRNA at w/w = 3/1, completely. While, PAMA-PMAMA-PEG
micelles binding siRNA needed higher w/w ratio, which was up
to 5/1. Lowest zeta potential of PAMA-PMAMA-PEG micelles
(Figure 5B) induced its weaker siRNA binding capacity. All the
results implied that the micelles formed by PAMA-PMsMA-
PEG could be used for further siRNA delivery in vitro. Further,
The particle sizes and zeta potentials of PEI/siRNA complexes
and micelles/siRNA micelleplexes were determined by DLS. As
shown in Figure S1A, Supporting Information, the sizes of PEI
and micelles after binding siRNA were 90–200 nm. Moreover,
the results in Figure S1B, Supporting Information, showed that
the zeta potentials were 7.5 ± 0.72, 15.8 ± 0.33, 11.4 ± 0.62, 9.6 ±
0.56, 7.7 ± 0.60, 5.2 ± 0.59 mV, respectively. All the results illus-
trated that the micelles were suitable for in vivo siRNA delivery.
41]
2
.4. Physicochemical Properties of PAMA-PMsMA-PEG Micelles
The obtained PAMA-PMsMA-PEG polycations were used to har-
vest cationic micelles. Normally, the polycations were dissolved
into trifluoroethanol and dropped added into PBS or deionized
water. The NPs were self-assembled through hydrophobic inter-
action. The sizes, zeta potentials, morphology, and distributions
were determined by laser particle size analyzer and TEM. As
shown in Figure 5A, the sizes of PAMA-PMMMA-PEG, PAMA-
PMEMA-PEG, PAMA-PMPMA-PEG, PAMA-PMBMA-PEG, and
PAMA-PMAMA-PEG micelles were about 247.7 ± 8.7, 99.3 ± 1.0,
123 ± 8.8, 158.3 ± 8.3, and 211.6 ± 11.9 nm, respectively. And the
TEM results in Figure 5C reflected all the polycations that could
form micelles, and the sizes of dry particle size were smaller
than that of hydrated particle size. However, the sizes of PAMA-
PMsMA-PEG NPs showed no regularity with the hydrophobic
sections changing. Notably, the PAMA-PMMMA-PEG and
PAMA-PMAMA-PEG micelles had the biggest sizes among the
five cationic micelles. For the PAMA-PMMMA-PEG, the bigger
sizes might be caused by the highest pKa values, which resulted
in the inattentive hydrophobic core. Nevertheless, the PAMA-
PMAMA-PEG with stronger steric hindrance in the hydro-
phobic sections caused the bigger sizes. The zeta potentials of
PAMA-PMMMA-PEG, PAMA-PMEMA-PEG, PAMA-PMPMA-
PEG, PAMA-PMBMA-PEG, and PAMA-PMAMA-PEG micelles
in Figure 5B were about 34.2 ± 1.6, 32.1 ± 0.1, 29.0 ± 0.5, 24.7 ±
2.5. Efficiencies of siRNA Transfection In Vitro
As known, an ideal siRNA delivery vehicle could not only
owe high siRNA delivery efficiency but also reduce cytotox-
icity. Herein, HepG2-Luc cells, which luciferase was stably
expressed, were used to evaluate the gene knockdown effi-
ciency. And the anti-firefly siRNA (siFL) was selected to silence
the luciferase protein. Moreover, polyethylenimine (PEI25k
)
1.6, and 21.5 ± 0.1 mV, respectively. It could be seen that the
was used as positive control. As shown in Figure 6A, except
for PAMA-PMBMA-PEG micelles, the level of luciferase pro-
tein decreased with the w/w ratios increasing. Excitingly,
zeta potentials declined monotonically with increasing the
length of alkyl substituent group on hydrophobic core. The nar-
Figure 6. Efficiencies of gene transfection in vitro. A) Efficiency of Luciferase knockdown on HepG2-Luc cells and B) cell viability at different w/w ratios
siRNA concentration: 50 nm). C) The luciferase knockdown efficiency of PAMA-PMEMA-PEG/siFL and PEI/siFL with different siRNA concentrations
on HepG2 cells (w/w = 10). Each bar represents Mean ± SEM of three experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 versus Blank.
(
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Figure 7. Cell uptake and endosome escape ability. A–B) Intracellular fluorescence intensities of PAMA-PMEMA-PEG NPs/Cy5-siRNA micelleplexes
determined following flow cytometry at w/w = 10/1. (C) Statistics of colocated ratios of Cy5-siRNA and LysoTracker Green. D) Intracellular distri-
bution of Cy5-siRNA determined by CLSM. (scale bar: 50 µm). Each bar represents Mean ± SEM of three experiments. *p < 0.05, **p < 0.01 and
*
**p < 0.001 versus Naked siRNA.
PAMA-PMEMA-PEG/siFL showed the same knockdown effect
at w/w = 10/1 with PEI/siFL at w/w = 15/1. Furthermore,
among the five vehicles, PAMA-PMEMA-PEG NPs showed
the best gene delivery efficiency at the same w/w ratio. The
luciferase protein knockdown efficiency might be related to
cytotoxicity. Therefore, the cytotoxicity test was carried out. As
shown in Figure 6B, there is no doubt that higher w/w ratio
resulted in higher cytotoxicity. However, higher cytotoxicity
PEI25k. Thus, it was concluded that PAMA-PMEMA-PEG
micelles could deliver siRNA effectively.
2.6. Cell Uptake and Endosomal Escape Ability
Commonly, the internalization and escape ability are regarded
as the two key factors for efficient siRNA delivery intracel-
[
42]
(
62.3%) was induced by PAMA-PMEMA-PEG/siRNA only up
lular. Therefore, to investigate the internalization and escape
ability, the FACS and CLSM were carried out. As mentioned
above, PAMA-PMEMA-PEG micelles showed the highest gene
silencing efficiency among the five cationic vehicles. And
PAMA-PMMMA-PEG also showed somewhat siFL delivery
efficiency. Therefore, the PAMA-PMMMA-PEG and PAMA-
PMEMA-PEG were chosen for further investigation. Cy5 labeled
to w/w = 15/1. Summary, PAMA-PMEMA-PEG NPs showed
efficient siRNA delivery without high cytotoxicity at w/w =
1
0. To further verify the effectiveness of PAMA-PMEMA-PEG
micelles, we investigated the luciferase knockdown effect
under different concentrations of siFL (w/w = 10). Figure 6C
showed that the luciferase knockdown effect showed con-
centration dependence, and the gene silencing efficiency of
PAMA-PMEMA-PEG/siFL was up to 81.9% with the concen-
trations of siFL was 50 nM. Moreover, under the same siFL
concentration, PAMA-PMEMA-PEG micelles showed better
luciferase protein silencing efficiency than positive control
siRNA was used as target gene, and polyethyleneimine (PEI25k
)
was chosen as positive control. As shown in Figure 7A,B, com-
pared with PAMA-PMMMA-PEG/Cy5-siRNA, PAMA-PMEMA-
PEG/Cy5-siRNA induced higher cell uptake efficacy. However,
the cell uptake efficacies of PAMA-PMMMA-PEG/Cy5-siRNA
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Figure 8. Cell apoptosis assay after treatment with siRNA-loaded PAMA-PMEMA-PEG micelles for 48 h on HepG2 cells (w/w = 10, siRNA: 50 nM).
[
41]
and PAMA-PMEMA-PEG/Cy5-siRNA were lower than PEI25k
Cy5-siRNA.
/
is related to resistance to chemotherapy, malignancy,[42] DNA
[
43]
[44]
replication,
and cellular invasion.
Therefore the RRM2
Further, endosomal escape abilities of PAMA-PMMMA-PEG
and PAMA-PMEMA-PEG were analyzed by CLSM (Figure 7D).
In Figure 7D, the signal of red represented the Cy5-siRNA
escaped into the cytoplasm, whereas the yellow signal
denoted the overlap of Lysottracker green and Cy5-siRNA.
The co-localization ratio in Figure 7C calculated by Figure 7D
showed PAMA-PMEMA-PEG owned higher escape ability
than PAMA-PMMMA-PEG, while, the escape ability of PEI/
Cy5-siRNA was higher than PAMA-PMEMA-PEG/Cy5-siRNA.
Although PAMA-PMEMA-PEG showed lower cell uptake and
escape ability than PEI25k, PAMA-PMEMA-PEG displayed
higher gene silencing efficiency at the same w/w ratio than
PEI25k. The reason for this phenomenon was that the form of
micelleplexes formed by polycationic micelles/gene could cause
higher gene silencing efficiency than PICs.[16] To verify the phe-
nomenon, Circular dichroism (CD) was carried out. As shown
in Figure S2, Supporting Information, the PEI showed greater
influence on the siRNA secondary structure and a red shift in
the signal was observed. The results suggest a much stronger
binding interaction formed with PEI, potentially distorting the
siRNA and/or inhibiting proper accessibility of the payload to
polymerase enzymes needed for effective transcription.
protein knockdown by siRRM2 could suppress the tumor. After
the HpeG2 cells were co-cultured with PAMA-PMEMA-PEG/
siRRM2 micelleplexes for 24 h, the cell apoptotic was deter-
mined. As shown in Figure 8, PAMA-PMEMA-PEG/siRRM2
micelleplexes resulted in the highest apoptotic and ratio of
late apoptotic up to 44.8%, which was much higher than PEI/
siRRM2 (31.2%). Moreover, either PAMA-PMEMA-PEG/siNC
or PAMA-PMEMA-PEG/siRRM2 micelleplexes did not induce
necrotic, which implied that PAMA-PMEMA-PEG/siRNA
micelleplexes had good biocompatibility. Above results in vitro
demonstrated that PAMA-PMEMA-PEG has great potential for
further use in the design of gene vehicles. Obviously, the non-
degradation induced the inherent toxicity need to be addressed.
However, the asymmetric structures of tertiary amine groups
introduced in gene vehicles provide multiple choices in the
design of polycationic carriers for gene delivery.
3
. Conclusion
In the presented work, a new series of asymmetric alkyl-sub-
stituted aminoethyl methacrylate were synthesized and used
as pH-sensitive hydrophobic monomers to prepare a library
of PAMA-PMsMA-PEG polycationic polymers as gene vehi-
cles. Their physicochemical properties such as buffering capa-
bility, siRNA binding ability, and in vitro cytotoxicity as well
as their complexation with siRNA in aqueous system were
also investigated. The results indicated that the asymmetric
alkyl substituents of the tertiary amine groups play a key role
in the proton buffering capacity of both monomers and corre-
sponding polycations. Increasing the length of alkyl substituent
groups decreased their buffering capacity and pKa values. More
2
.7. Anti-Tumor Cell In Vitro of PAMA-PMEMA-PEG/siRRM2
Micelleplexes
To investigate the in vitro anti-tumor efficiency of PAMA-
PMEMA-PEG micelles, the apoptosis experiment was per-
formed. PEI25k was used as the positive control, and siRRM2
was chosen as the targeted gene. Human tumors could overex-
press M2 subunit of ribonucleotide reductase (RRM2), which
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importantly, PAMA-PMEMA-PEG with pKa about 6.2 not only
can exhibit high siRNA transfection efficiency and low cytotox-
icity, but also can undergo protonation in the endosomal acidic
environment, leading to efficient endosomal escape and consid-
erable anti-tumor efficiency in vitro. It is expected that PAMA-
PMEMA-PEG can be applied as promising carriers for efficient
siRNA delivery.
and AIBN (1.57 mg, 0.01 mmol) were dissolved in DMF (3 mL). The
pretreatment, polymerization, and post-treatment were same as the
procedure mentioned above. And then the obtained PBAMA-PMEMA
was used as macro-RAFT agent to polymerize with poly(ethylene glycol
methacrylate) at molar ratio of 1:4 to obtain PBAMA-PMEMA-PEG
under same condition. Finally, The PAMA-PMEMA-PEG was obtained
by deprotection by TFA. Other PAMA-PMsMA-PEG polymers were
synthesized by same procedure except for the MsMA. All the polycations
1
were stored and characterized by H NMR and GPC for further usage.
The molecular weights and polydispersity index (PDI) of the polymers
were measured with a Waters 1515 gel permeation chromatographer
4
. Experimental Section
(
GPC, Waters company, Milford, USA) equipped with refractive index
Materials:
-(dimethylamino)ethyl methacrylate (DMAEMA), azobisisobutyronitrile
AIBN), bromoethane, 1-bromopropane, 1-bromobutane,
-bromopentane, acetonitrile (AN), dimethyl sulfoxide (DMSO), N,N-
N-methylethanolamine,
methacryloyl
chloride,
detector, using polystyrene as the standard material for calibration and
−
1 1
2
DMF as the eluent at a flow rate of 1 mL min . H NMR spectra of
MsMA and PAMA-PMsMA-PEG were recorded on a Varian Inova-500M
instrument (Varian Inc., Palo Alto, USA) with CDCl3 or d-DMSO as a
solvent and tetramethylsilane (TMS) as the internal standard.
(
1
dimethylformamide (DMF), tetrahydrofuran (THF), triethylamine
(
(
TEA), dichloromethane (DCM), ethyl acetate (EA), petroleum ether
PE), diethyl ether, and trifluoroacetic acid (TFA) were obtained from
Preparation of PAMA-PMsMA-PEG Micelles: Typically, 10 mg PAMA-
PMsMA-PEG was dissolved in 1 mL trifluoroethanol and drop added
into deionized water (10 mL). Waiting for the trifluoroethanol to
Jiangtian Chemical Reagents Co., Ltd. (Tianjin, China). Positive control
polyethylenimine 25 kDa (Gibco, USA), 2-(methylamino) ethanol, and
poly(ethylene glycol methacrylate) (EG) with molecular weight of 500
were purchased from aladdin (Shanghai, China). N-(tert-Butoxycarbonyl)
aminoethyl methacrylate (BAMA) was synthesized according to the
−
1
evaporate, the nanoparticles concentration was adjusted to 1 mg mL
for further usage. The zeta potentials and sizes of PAMA-PMsMA-PEG
nanoparticles and micelles/siRNA micelleplexes were determined by
dynamic light scattering (Zetasizer Nano ZS, Malvern, UK).
[
23]
previous study.
Determination of siRNA Binding Capacities: Agarose gel retardation
assay was used to determine the siRNA binding capacity. PAMA-
PMsMA-PEG nanoparticles and siRNA (50 nM) with w/w ratios of 1/1,
3/1, 5/1, and 10/1 were incubated at room temperature for 20 min, and
mixed with 3 µL 6× loading buffer. And then, the volume was adjusted to
20 µL. Finally, the solution was added into agarose gel hole (2%), which
contained ethidium bromide (5 µL). The electrophoresis was carried out
under 120 V for 20 min in 1× TAE running buffer. Finally, the gel was
characterized at UV light (254 nm, VDS thermal imaging system).
Acid–Base Titrations of MsMA and Tri-Block Polycations: The
pH-sensitive abilities of MsMA and tri-block polycations were carried out
by acid–base titrations. In a word, 40 mg materials dissolved in 0.1 M
HCl (3 mL). After that, the volume was adjusted to 20 mL. And then,
NaOH solution (0.1 M) was drop added. The pH values and NaOH
consumption volumes were recorded respectively.
Luciferase Assay: HepG2-Luc cells, which expressed luciferase protein
stably, were plated in 24-well plates for 24 h. Then the medium changed
with Opti-MEM (0.5 mL), the PAMA-PMsMA-PEG/siFL (siRNA: 50 nM)
micelleplexes with different w/w ratios were added, respectively. After
transfecting for 4 h, the medium was replaced with DMEM (1 mL) and
cultured for another 20 h. After that, HepG2-Luc cells were washed
with PBS and lysed with passive lysis buffer (100 µL) and made sure
the cells lysis completely. Finally, centrifuged for 1 min at 12 000 rpm,
and collected the supernatant for luminescence measurements by Multi-
Mode Microplate Reader (Synergy HT, BioTek, USA).
Tris Base, Ethidium bromide, Dulbecco's modified Eagle's medium
DMEM), fetal bovine serum, trypsin, Opti-MEM, and Penicillin-
(
streptomycin were bought from Invitrogen Corporation. Cy5-labeled
siRNA (Cy5-siRNA), negative controlled siRNA (siNC), anti-firefly siRNA
(
siFL), and anti-RRM2 siRNA (siRRM2) were provided by Suzhou Ribo
Life Science Co., Ltd. (Suzhou, Jiangsu Province, China).
Synthesis of N-Methyl-N-Alkyl Aminoethyl Methacrylate (MsMA): The
asymmetric alkyl-substituted aminoethyl methacrylate monomers were
synthesized by nucleophilic substitution and esterification reaction.
Taking N-methyl-N-ethyl aminoethyl methacrylate (MEMA) as an
example, N-methyl ethanolamine (15.022 g, 0.2 mol), 1-bromoethane
(
24 g, 0.22 mol), and anhydrous sodium carbonate (26.5 g, 0.25 mol)
were dissolved into 400 mL acetonitrile. After the reaction at 40 °C in an
oil bath for 8 h, anhydrous sodium carbonate was removed by filtration
and acetonitrile was removed by rotary evaporation. The raw product was
washed with saturated sodium chloride solution three times, extracted
with ethyl acetate to obtain N-methyl-N-ethyl ethanolamine. And then,
N-methyl-N-ethyl ethanolamine (5.3 g) and trimethylamine (7.8 g,
7
7 mmol) were dissolved in THF (50 mL) and cooled to 0 °C in ice-water
bath. And then methacryloyl chloride was added slowly to the mixture.
After reacting for 12 h, MEMA was separated by column chromatography
(
EA/PE = 2/5). The yield for MEMA was 80%. Other monomers were
prepared by similar procedure except for the bromoalkyl. All the
1
monomers were characterized by H NMR and stored for further usage.
The siRNA sequences were listed in Table S1, Supporting Information.
Synthesis and Characterizations of PAMA-PMsMA-PEG: The tri-block
PAMA-PMsMA-PEG polymers were synthesized by a tri-step sequential
RAFT polymerization. The first block amino-protected poly[N-(tert-
butoxycarbonyl) aminoethyl methacrylate](PBAMA) with pre-ordained
polymerization degree of about 50 was prepared in the presence of
S-Dodecyl-S′-(α,α′-dimethyl-α″-acetic acid)-trithiocarbonate as RAFT
agent and AIBN as initiator in DMF. Typically, RAFT agent (95.6 mg
Cell Viability: HepG2 cells were incubated in 96-well for 24 h. Cells
were treated with PAMA-PMsMA-PEG/siRNA micelleplexes for 24 h.
Then, cells were washed with PBS three times and the medium was
replaced with fresh DMEM medium containing 10 µL CCK-8. After
20 min, the absorbance of medium was determined by Multi-Mode
Microplate Reader.
Confocal Laser Scanning Observation: The intracellular distribution
of PAMA-PMsMA-PEG/Cy5-siRNA complexes was determined by
laser scanning confocal microscope. In summary, HepG2 cells were
plated into 35 mm dishes for 24 h. And cells were treated with PAMA-
PMsMA-PEG/Cy5-siRNA micelleplexes for 4 h. And then, cells were
washed with PBS three times and stained with Lysotracker Green
(staining endosomes and lysosome) and DPAI (staining nuclei). At last,
Zeiss confocal microscope (LSM700, Carl Zeiss, Germany) was used to
image cells.
0
.26 mmol), N-(tert-butoxycarbonyl) aminoethyl methacrylate (3 g,
13.1 mmol) and initiator AIBN (3.28 mg, 0.02 mmol) were dissolved in
DMF (5 mL). After complete dissolution and three freeze-pump-thaw
cycles, the flask was placed in a thermostatic water bath at 70 °C for
about 24 h under Argon atmosphere. The PBAMA was isolated and
purified by dialysis and lyophilization, and then was used as a macro-
RAFT agent in the subsequent polymerization to prepare PBAMA-
PMsMA with pre-ordained polymerization degree of about 100 and
PBAMA-b-PMsMA-PEG. After deprotection by TFA, the polycations
PAMA-PMsMA-PEG were obtained. Taking PAMA-PMEMA-PEG as
an example, PBAMA (0.51 g, 0.05 mmol), MEMA (0.85 g, 5 mmol),
5
Flow Cytometry Detection: HepG2 cells (1 × 10 ) were seeded into
6-well plate, and cultured for 24 h. Then the medium was replaced with
Opti-MEM, and the PEI/Cy5-siRNA and PAMA-PMsMA-PEG/Cy5-siRNA
complexes (w/w = 10/1) were added to culture 4 h. Finally, the tumor
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cells were digested and washed three times with PBS. Fast test was
administrated under FACS Caliber flow cytometry (Becton Dickinson,
San Jose, CA, USA).
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Apoptosis Analysis: HepG2 cells (1 × 10 ) were incubated in 6-well
plate for 24 h. Further, the cells were treated with PBS, siRRM2, PEI/
siNC, PEI/siRRM2, PAMA-PMEMA-PEG/siNC, and PAMA-PMEMA-PEG/
siRRM2 for 24 h (w/w = 10/1, siRNA: 50 nM). The cells were digested
and washed with PBS three times, as well as stained with PI and Annexin
V-FITC. Fast characterization was carried out under FACS Caliber flow
cytometry (Becton Dickinson, San Jose, CA, USA).
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