Structural exploration of hydrophobic core in polycationic micelles for improving siRNA delivery efficiency and cell viability

Article abstract of DOI:10.1039/c8tb02706d

Improving siRNA delivery efficiency often encounters a dilemma with poor or decreased biocompatibility for polycationic micelles. To address this dilemma, this work focused on a structural exploration of the hydrophobic core in amphiphilic polycationic micelles by preparing two amphiphilic polycations with block or random hydrophobic segments, poly(ethylene glycol)-block-poly(aminoethyl methacrylate)-block-poly(2-diamylamine ethyl methacrylate)-block-poly(2-diethylamine ethyl methacrylate) (mPEG-PAMA-PD5A-PDEA, PADE) and poly(ethylene glycol)-block-poly(aminoethyl methacrylate)-block-poly(2-diamylamine ethyl methacrylate-co-2-diethylamine ethyl methacrylate) (mPEG-PAMA-P(D5A/DEA), PA(D/E)). The properties of the two copolymers and their self-assembly micelles were characterized, including structure, morphology, size and zeta potential. Cytotoxicity, siRNA silencing efficiency and cellular uptake of PADE/siRNA and PA(D/E)/siRNA complexes were evaluated in HepG2 and MDA-MB-231 cells in vitro. The endosome escape and intracellular distribution of PADE/siRNA and PA(D/E)/siRNA in HepG2 cells were also observed by CLSM. Significantly, the results indicated that PA(D/E)/siRNA showed not only better gene silencing efficiency but also lower cytotoxicity, which may be attributed to the homogeneous morphology of the hydrophobic core of PA(D/E) micelles. Therefore, this work provides a new pathway to overcome the dilemma between siRNA delivery efficiency and biocompatibility for the development of efficient polycation carriers.

Full text of DOI:10.1039/c8tb02706d

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Guoping Chen et al.
Regulating the stemness of mesenchymal stem cells by tuning
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JPolueransael odfoMnaotteraiadljsuCsthemmairsgtrinysB
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DOI: 10.1039/C8TB02706D
Journal Name
ARTICLE
Structural Exploration of Hydropbobic Core in Polycationic Micelles
for Improving siRNA Delivery Efficiency and Cell Viability
Wenjun Huang±a,c, , Xiaoxia Wang±b, Changrong Wanga, Lili Dub, Jianhua Zhanga, Liandong Denga,
Huiqing Caob, Anjie Dong*a,c
Improving siRNA delivery efficiency often encounters a dilemma with poor or decreased biocompatibility for polycationic
micelles. To address the dilemma, this work focused on the structural exploration of hydrophobic core in amphiphilic
polycationic micelles by preparing two amphiphilic polycations with block or random hydrophobic segments, poly(ethylene
glycol)-block-poly(aminoethyl methacrylate)-block-poly(2-diamylamine ethyl methacrylate)-block-poly(2-diethylamine
ethyl methacrylate (mPEG-PAMA-PD5A-PDEA, PADE) and poly(ethylene glycol)-block-poly(aminoethyl methacrylate)-block-
poly(2-diamylamine ethyl methacrylate-co-2-diethylamine ethyl methacrylate) (mPEG-PAMA-P(D5A/DEA), PA(D/E)). The
properties of the two copolymers and their self-assembly micelles were characterized including structure, morphology, size
and zeta potential. Cytotoxicity, siRNA silencing efficiency and cellular uptake of PADE/siRNA and PA(D/E)/siRNA complexes
were evaluated in HepG2 and MDA-MB-231 cells in vitro. The endosome escape and intracellular distribution of PADE/siRNA
and PA(D/E)/siRNA in HepG2 cells were also observed by CLSM. Significantly, the results indicated that PA(D/E)/siRNA
showed not only better gene silencing efficiency but also lower cytotoxicity, which may be attributed to the homogeneous
morphology of the hydrophobic core of PA(D/E) micelles. Therefore, this work provided a new pathway to overcome the
dilemma between siRNA delivery efficiency and biocompatibility for development of efficient polycation carriers.
of low transduction efficiency yet. Beside cationic liposome,
various of polycationic based vectors have been prepared to
delivery siRNA, such as polyethylenimine (PEI) and its
Introduction
It's worth noting that gene therapy based on small interfering
RNA (siRNA) technology is a promising treatment for many kinds
of diseases,1-5 which attracts more and more attention.
However, naked siRNA in vivo is easily degraded by nucleic acid
enzyme, with high renal clearance rate and low endocytosis.6,7
Hence, a key strategy of gene therapy is to select the
appropriate gene carrier and gene introduction method, so that
the safe, efficient, controllable and stable expression of the
target gene in target cell could be obtained.8,9 However, gene
delivery has been encountered a dilemma between high
transfection efficiency and good biocompatibility.
At present, the gene carriers mainly include virus vectors
and non-viral vectors. Although with high transduction
efficiency, viral vectors are limited for application due to
immunogenicity and thus the safety concern and difficulties in
preparation.10,11 Non-viral vectors have the advantages of low
toxicity and low immunogenicity,12-15 but with the disadvantage
derivatives,16-19 polyamidoamine dendrimer,20, 21 poly(L-lysine)
(PLL)22,23 and poly(2-dimethylaminoethyl methacrylate)
(PDMA),24-29 etc., because they can interact with siRNA to form
nano-complexes, have structural flexibility and are easy to
prepare. To balancing the biocompatibility and transduction
efficiency, PEGylated (A segments) and hydrophobized (C
moieties) strategies have been widely used to modify
polycation (B moieties) to construct A−B−C, A−C−B, or A−(B/C)
types of amphiphilic polycations for core-shell micelles,30-37
which not only stabilized the siRNA-loaded complexes but also
improved the cell uptake, resulting in high improvement in
gene-silencing efficiency.
Our previous work had certificated A-B-C type of tri-block
copolymer showed more promising gene delivery efficiency
than A−C−B and A−(B/C).38 Recently, we studied a series of
triblock cationic copolymers, PEG block poly (aminoethyl
methacrylate) (PAMA) block pH-sensitive hydrophobic
segments with different pKa, and a significant discovery was
obtained that the better gene silencing efficiency was located in
a narrow range of pKa 5.8-6.2, which was contributed from
higher endosomal escape.39 Continuously, in this work as shown
in Scheme 1, we further explored the structural contribution of
the pH sensitive hydrophobic cores (pKa~6.0) with block or
random monomer units of 2-(diethylamine) ethyl methacrylate
(DEA) and 2-(diamylamine) ethyl methacrylate (D5A). Two kinds
a.
Department of Polymer Science and Technology, School of Chemical
Engineering and Technology, Key Laboratory of Systems Bioengineering (Ministry
of Education), Tianjin University, Tianjin 300072, China.
b.
Laboratory of Nucleic Acid Technology, Institute of Molecular Medicine, Peking
University, Beijing 100871, China.
c.
Collaborative Innovation Center of Chemical Science and Engineering (Tianjin),
Tianjin 300072, China.
± Wenjun Huang and Xiaoxia Wang contributed equally to this work.
*Email - ajdong@tju.edu.cn
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of tri-block copolymer mPEG2k-PAMA-P (D5A/DEA) (PA(D/E)) The polymer of mPEG2k-b-PAEMA was synthesized by a
View Article Online
DOI: 10.1039/C8TB02706D
and four-block polymer mPEG2k-PAMA-PD5A-PDEA (PADE) reversible addition-fragmentation chain transfer (RAFT)
were synthesized by RAFT polymerization. The molecular and polymerization of AEMA monomers using AIBN as the initiator.
micellar structures of PA(D/E) and PADE had been Firstly, the RAFT agent was prepared and subsequently the
characterized, and the intracellular siRNA delivery capacity and mPEG-based macro-RAFT agent (mPEG2k-DDMAT) was
cell viability had been evaluated. The results showed that the synthesized by esterification reaction of mPEG2k and S-dodecyl-
homogeneous composition and morphology of the inner core in S’-(,’-dimethyl-”-acetic
acid)-trithiocarbonate
PA(D/E) micelles facilitated both higher intracellular gene (DDMAT).41,42 Typically, AIBN, mPEG2k-DDMAT and AEMA were
silencing efficiency and better cell viability, which will help to added into the Schlenk tube with 2 mL of dimethylformamide
provide some reasonable suggestions in designing siRNA (DMF). The polymerization reaction was achieved at 70 °C for
carriers.
24 h, then the mixture was dialyzed for 48 h in double distilled
water and freeze-dried to get the polymer.
Experimental
Materials
Synthesis of mPEG2k-b-PAEMA-b-PD5A-b-PDEA (PAEDE) and
mPEG2k-b-PAEMA-b-P(D5A/DEA) (PAED/E)
The tri-block polymer PAEDE and four-block polymer PAE(D/E)
were synthesized by a method of RAFT polymerization as shown
in Fig. 1. The former adopted the method of adding monomers
in a certain order, the latter took a method of adding two
monomers together. Briefly, the polymerization reaction was
achieved at 70 °C for 24 h. Then the mixture was dialyzed for 48
h in double distilled water and freeze-dried to get the
copolymers.
Methoxy PEG (mPEG2k), azobisisobutyronitrile (AIBN), di-tert-
butyl dicarbonate (Boc), 2-aminoethanol, methacryloyl
chloride, 1-bromopentane, acetonitrile, dimethyl sulfoxide
(DMSO), TRIzol Reagent and 3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide (MTT) were purchased from
SigmaAldrich. Agarose was bought from GEN TECH (Hong Kong,
China). Lipofectamine 2000, Dulbecco’s modified Eagle’s
medium (DMEM), Opti-MEM, penicillin−streptomycin, and
trypsin were purchased from Thermo Fisher. Cy5-labeled siRNA
(Cy5-siRNA), siFL, and siRRM2 were supplied by Suzhou Ribo Life
Science Co., Ltd. (Jiangsu, China).
Synthesis of mPEG2k-PAMA-PD5A-PDEA (PADE) and mPEG2k-
PAMA-P(D5A/DEA) (PA(D/E))
Trifluoroacetic acid (TFA) was used as a deprotection agent to
deprotect the boc-protection on PAEDE and PAE(D/E). Then the
deprotected copolymers were dialyzed for 48 hours in double
distilled water and freeze-dried to get the copolymers PADE and
PA(D/E). The copolymers were characterized using H NMR.
(Varian Unity Plus INOVA 400).
Synthesis of 2-(diamyl amine) ethyl methacrylate (D5A)
Ethanolamine and 1-bromopentane were added into a three-
round bottom flask with acetonitrile. In addition, Na2CO3 was
added into reaction bottle. The reaction mixture was stirred at
1
o
80 C for 12 h. The reaction mixture was then filtered and
evaporated. The condensed product was dissolved in
tetrahydrofuran and added with triethylamine. After that,
methylacryloyl chloride was added slowly into the reaction
bottle at 0 C. After 12 hours of reaction, the mixed reactant
was filtered and evaporated to obtain the crude product, and
the product was finally obtained by vacuum distillation.
Preparation and characterization of PADE and PA(D/E) micelles
10 mg PADE or PA(D/E) were dissolved in 1 mL trifluoroethanol
(TFE) and the obtained solution was added dropwise slowly into
5 mL double-distilled water under stirring. Then the solution
was dialyzed against deionized water for two days, completely
removing TFE and obtaining PADE or PA(D/E) micelles. The
dynamic laser scattering (Zetasizer Nano ZS, Malvern, UK) was
used to measure the size distribution and zeta potential of the
micelles. In addition, transmission electron microscopy (TEM,
JEM-100CX, Philips EM400ST) was used to observe the
morphologies of the micelles.
o
Synthesis of N-(tert-Butoxycarbonyl) aminoethyl methacrylate
(AEMA)
According to previous reports,40 N-(tert-butoxycarbonyl) amino
ethyl methacrylate was prepared. Tert-butyl N-(2-hydroxyethyl)
carbamate (10 g, 62 mmol) and triethylamine (12.9 mL, 93
mmol) were added to the reaction bottle containing CH2Cl2 (100
mL), then methacrylate was added dropwise slowly to the
Measurements of pKa values
o
The acid-base titration method43 was performed to test the pKa
values of PADE and PA(D/E). Briefly, the copolymer was first
dissolved in HCl (0.1 M), and then added 50 L NaOH (0.1 M)
each time under vigorous stirring. The Mettler Toledo pH meter
with microelectrode was used to detect the pH changes. pKa
was defined as the pH value between the two equivalent points
in the titration curve.
mixture at 0 C with Ar conditions. After 1 h, the reaction
mixture was transferred to room temperature and continued to
react for 12 h. The reaction mixture was washed with 10% citric
acid, 10% HCl, 10% K2CO3, sat NaHCO3 and brine, respectively.
After that, the solution was dried by MgSO4 and removed the
solvent under reduced pressure. Finally, the desired product
was purified by recrystallization using
dichloromethane and n-hexane (v/v = 1/9).
a mixture of
Differential Scanning Calorimeter (DSC) measurements
Synthesis of mPEG2k-b-PAEMA(PAE)
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The DSC measurements were performed by using the Netzsch efficiency of mRNA level. Briefly, the HepG2 cells were plated in
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5
DOI: 10.1039/C8TB02706D
DSC 200 F3 under a constant flow of nitrogen of 50 mL/min. The 6-well plates (2 × 10 cells each well) for 24 h, the PADE/siRRM2
freeze-dried powder of PADE or PA(D/E) micelles were used to and PA(D/E)/siRRM2 complexes were used to transfect cells and
explore the crystallization of PADE or PA(D/E) micelles. 10 mg then incubated for 24 h. The total RNA was extracted using a
sample was added into a Tzero aluminum hermetic DSC pan. TRIzol Reagent (Thermo Fisher) and converted to cDNA by
The sample pan was placed into the DSC well, which was cooled reverse transcription. Then, the RT-PCR assay was performed by
to 0 °C in advance. In the experiment, the DSC curve was using a SYBR Green PCR Master Mix with GAPDH as the internal
recorded from 0 °C to 300 °C at a rate of 5 °C/min.
control.
The pH-sensitivity assembly/disassembly behavior.
Cell viability
The pH-sensitivity of polymer micelles was evaluated by Nile red MTT assay was carried out to evaluate the cytotoxicity of
based fluorescent probe method. In brief, the 20 g of Nile red PADE/siRNA and PA(D/E)/siRNA complexes. After the HepG2
dye was encapsulated into PADE or PA(D/E) micelles (1 mg/mL) cells or were seeded in 96-well plates (1 × 104 cells per well) for
by hydrophobic interaction, which was used to measure the 24 h, the siRNA-loaded copolymer micelles (N/P = 5, 10, and 15;
micellar assembly/disassembly states by track the fluorescent siNC dose: 30 nM, 50 nM) were used to treat the cells for 24 h,
signal of Nile red dye. The pH of the Nile red loaded micelle after incubation for 24 h, 10 µL of MTT solution with a
solution was subsequently regulated to 7.4, 7.2, 7.0, 6.8, 6.5, concentration of 5 mg/mL in PBS was added in each well and
6.2, 6.0, 5.8, 5.6, 5.2, 4.5, 4.0, 3.5, 3.0 and 2.4. After 2 h, the incubated for 4 h. Then, the medium was replaced with DMSO
Varian fluorescence spectrophotometer (Varian, USA) was used (50 µL/well) to dissolve the formazan crystals. The Multi-Mode
to measure the fluorescent value of the micelles solution.
Microplate Reader was used to measure the absorbance at 540
nm (650 nm used as the reference wavelength). The untreated
cells were used as controls.
Preparation of siRNA-loaded complexes
The PADE or PA(D/E) micelles/siRNA complex was prepared by
electrostatic self-assembly. The copolymer micelle dispersion
In virto cellular uptake and cellular distribution
was mixed with the same volume of siRNA solution, and The HepG2 cells and MDA-MB-231 cells were used. The cells
repeatedly blow several times or high speed concussion 5 s, and plated in 6-well plates (2 × 105 cells each well) for 24 h before
then incubate for 20 min at room temperature for using.
transfection for cellular uptake studies. After 24 h incubation,
the Cy5-siRNA-loaded copolymer micelles (N/P = 5, 10; Cy5-
siRNA: 30 nM, 50 nM) were used to treat the cells for 4 h at 37
°C. Then, remove the culture medium and wash it three times
with 1 mL PBS to remove the residual free complexes, and
followed by suspended in PBS. The fluorescence signal was then
detected by a FACS Calibur flow cytometer (Becton Dickinson,
San Jose, CA, USA).
The cells were seeded in 35 mm dishes with 2 × 105 cells
each well for 24 h to evaluate the cellular distribution. After 24
h, the cells were treated with the PADE/Cy5-siRNA and
PA(D/E)/Cy5-siRNA (N/P = 5, 10, and 15; siRNA dose: 30 nM, 50
nM) and incubated for 4 h. After incubation, remove the liquid
and wash with cold PBS for 3 times. Subsequently, DAPI was
used to mark cell nuclei and Lysotracker Green was used to
mark endosome/lysosome. After the staining, the cells were
washed with PBS for 3 times, and Zeiss confocal microscope
(LSM700, Carl Zeiss, Germany) was used to observe the
fluorescence and take photos.
Agarose gel electrophoresis assay
The agarose gel retardation assay was carried out as follows: 20
µL copolymer micelles (CMs)/siRNA complex solution (N/P = 2,
5, 8, 10) was fully mixed with 4 µL of 6 x loading buffer (Takara
Biotechnology, Dalian, Liaoning Province, China), the mixture
was then totally loaded onto 2% agarose gel containing 5 µg/mL
ethidium bromide. Electrophoresis was carried out for 30 min
at 120 V voltage when the electrophoresis buffer reached 1 ×
TAE. At last, the results of the siRNA electrophoresis were
viewed at UV light wavelength of 254 nm with an image master
VDS thermal imaging system (Bio-Rad, Hercules, CA).
In vitro gene silencing
To evaluate the gene silencing ability of copolymer
micelles/siFL, the MDA-MB-231-Luc cells and HepG2-Luc cells
were plated in 24-well plates with 5 × 104 cells each well for 24
h. Subsequently, the medium was removed and added Opti-
MEM (600 μL) containing 0.4 g copolymer micelles/siFL (N/P =5,
10, 15). After 24 h incubation, the culture medium was removed
and the cells were washed with PBS for three times. Then, the
cells were lysed using 100 µL of 1 × passive lysis buffer (Promega
Co., Madison, WI) for 30 min and accompanied by violent
shaking to ensure complete lysis. After that, the cell lysate was
centrifuged and the fluorometer (Synergy HT, BioTek, USA) was
used to measure the relative luminescence intensity.
Results and discussion
Preparation and characterization of PADE and PA(D/E)
Firstly, the monomer 2-(diamyl amine) ethyl methacrylate
(D5A-MA) was prepared. The four-block copolymers PADE was
prepared by sequential RAFT of N-(tert-butoxycarbonyl)
aminoethyl methacrylate (AEMA), 2-(diethylamine) ethyl
methacrylate (D5A), and 2-(diamyl amine) ethyl methacrylate
(DEA) monomers. Difference from the four-block polymer,
PA(D/E) was prepared by adding the mixture of two monomers
The reverse transcription polymerase chain reaction (RT-
PCR) assay was performed to evaluate the gene-silencing
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in the second step of RAFT polymerization (Fig. 1). The PDE) and poly(ethylene glycol)-block-poly(2-diamylamine ethyl
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DOI: 10.1039/C8TB02706D
structures of D5A-MA and the intermediate products PAE, PAED, methacrylate-co-2-diethylamine ethyl methacrylate) (mPEG-
PAEDE, PAE(D/E) were confirmed by the proton NMR (1H NMR) P(D5A/DEA), P(D/E)). The 1H NMR spectra were taken to
spectra (Fig. 2A). The disappearance of the peaks at b3 and b5 characterize the structure of PDE and P(D/E), and the zeta
(Fig. 2B and 2C) confirmed PADE and PA(D/E) were synthesized potentials of PDE and P(D/E) micelles were evaluated(shown in
successfully. The degree of polymerization of each component Fig. S1A and B). P(D/E) micelles with slightly higher zeta
1
of the two polymers was calculated by H NMR, labeled as potential(16mV) than that of PDE(12mV). The pKa values of DEA
mPEG45-PAMA30-PD5A31-PDEA36 (PADE) and mPEG45-PAMA30- and D5A are about 8.5 and 5.0, respectively, which means DEA
P[D5A29/DEA29] (PA(D/E)). Then the molecular weight could be moieties are facile to be charged positively in the testing
calculated as shown in Table 1. In addition, the gel permeation medium of double-distilled water (pH»7.0 ). The higher zeta
chromatography was used to measure the polydispersity index potential of P(D/E) micelles indicated that there are more
of PADE and PA(D/E) copolymers, as listed in Table1.
positive charged DEA moieties on the core-shell interface of
P(D/E) micelles due to the random core structure than that of
PDE micelles with block core structure. Therefore, it could be
thought that the higher zeta potential of the random structural
core(D/E) lead to the higher zeta potential of PA(D/E).
Characterization of PADE and PA(D/E) micelles
As amphiphilic copolymers, PADE and PA(D/E) could self-
assemble into micelles, which could bind siRNA by electrostatic
interactions. Dynamic light scattering (DLS) and transmission
electron microscopy (TEM) were used to determine the size
distribution and morphology of PADE and PA(D/E) micelles,
respectively. As showed in Fig. 3, PADE and PA(D/E) micelles all
possessed spherical structures with similar size about 90 nm
and narrow particle size distributions. The acid-base titration
curves (Fig. 4A) certificated that PADE and PA(D/E) had similar
pKa6.0, but PADE showed two transitions at pH 4.0 and 6.8,
supposing to be leaded by the block structure of PD5A and PDEA
segments. Hydrophobic Nile red dye was used as the
fluorescent probe encapsulated into the micellar hydrophobic
core to track the pH sensitive assembly/disassembly behavior.
As shown in Fig. 4B and 4C, the fluorescent signal of Nile red dye
weakened gradually with pH decreasing and became
undetectable, indicating the hydrophobic environment of the
core gradually disappeared due to micellar disassembly with the
protonation of DEA and D5A moieties in the core. Also,
different from PA(D/E) micelles with a continuous variation in
fluorescent signal, PA(D/E) micelles showed two steps of
decrease, indicting two phases maybe exist in PA(D/E) micellar
cores. The further evaluation results of PADE and PA(D/E)
micelles by DSC were shown in Fig. 4D. The DSC curve of PADE
In vitro gene silencing efficiency and internalization
The luciferase assay was performed to estimate the capacity of
PADE and PA(D/E) micelles in siRNA delivery in the MDA-MB-
231-Luc cells and HepG2-Luc cells. As shown in Fig. 5A, in MDA-
MB-231-Luc cells, PA(D/E)/siRNA complex showed higher gene
silencing efficiency at 24 h than PADE/siRNA complex at the
same N/P ratios and it was worth noticing that all the gene
silencing efficiencies of PA(D/E)/siRNA complex at N/P = 5, 10,
15 were higher than lipo 2000. Importantly, PA(D/E)/siRNA
complex could maintain the more dominance in gene silencing
efficiency after 48 hours (Fig. 5B). In HepG2-Luc cells, as shown
in Fig. 5C and 5D, PA(D/E)/siRNA and PADE/siRNA showed the
similar luciferase silencing effects at 24 h or 48 h, which were
also better than Lipo 2000 at N/P = 15. In addition, it could be
seen that PA(D/E)/siRNA presented higher gene silencing
efficiency in MDA-MB-231-Luc cells than HepG2-Luc cells.
To further confirm the gene delivery capacities of
PA(D/E)/siRNA and PADE/siRNA, RT-PCR assay were carried out
by using siRRM2. Ribonucleotide reductase M2 (RRM2) has
great significance for DNA synthesis and cell growth control,
which has made it an important target for cancer treatment.44
As shown in Fig. 6A and B, both at 30nm and 50 nm doses of
siRNA, PA(D/E)/siRRM2 obtained higher knockdown efficiencies
in the RRM2 mRNA expression than PADE/siRRM2 at N/P = 10
and N/P = 15, which reached the level of Lipo 2000.
After 4 h incubation, fluorescence-activated cell sorting
(FACS) was performed to evaluate the percentage of the cells
that internalized Cy5-siRNA-loaded micelles (N/P = 5, 10) (Fig. 6
C,D,E,F). The results of mean fluorescence intensity in HepG2
cells (Fig. 6 C,D) clearly indicated that Cy5-siRNA-loaded PADE
and PA(D/E) micelles showed similar cell uptake efficiencies at
N/P = 5, however, the PA(D/E) showed a higher uptake
efficiency than PADE at N/P = 10. In MDA-MB-231 cells (Fig. 6
E,F), obviously higher cell uptake levels of PA(D/E)/siRNA than
PADE/siRNA were observed at different N/P ratios. The higher
cell uptake of PA(D/E)/siRNA was related to high zeta potentials
of PA(D/E) than PADE micelles as shown in Fig. 4A, resulting in
higher gene silence efficiency of PA(D/E)/siRNA. Moreover,
from the cell uptake results in Fig. 6(C,D,E,F), with Lipo 2000 as
control, it could be seen that both PA(D/E)/siRNA and PADE
o
exhibited two melting peaks at 150 oC and 225 C, assigned to
the PD5A and PDEA crystalline phases, respectively. For
PA(D/E), only one melting peak at about 215 oC was observed.
Therefore, it could be thought the hydrophobic core of PA(D/E)
micelle took a homogeneous morphology and that of PADE
micelle took microphase separation structure as shown in
Scheme 1. Besides, PA(D/E) micelles showed slightly higher zeta
potential (34 mV) than that of PADE (29 mV) (Fig. 4E), while they
possessed similar siRNA bonding capacity completely at a N/P
ratio of 5.0 (Fig. 4F).
About the difference of zeta potential between PA(D/E) and
PADE, we speculated that the structure difference of
hydrophobic cores between PA(D/E) and PADE micelles may
attribute to the difference in zeta potential. To certificate this
speculation, we synthesized two kinds of copolymers without
the cationic block by RAFT polymerization, poly(ethylene
glycol)-block-poly(2-diamylamine ethyl methacrylate)-block-
poly(2-diethylamine ethyl methacrylate (mPEG-PD5A-PDEA,
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/siRNA showed obviously higher cell uptake levels in MDA-MB- also lower cytotoxicity than PADE. We thought the advantage
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231 cells than HepG2 cells, and more higher level of cell uptake properties of PA(D/E) micelles may be attributed to the
for PA(D/E)/siRNA than PADE /siRNA showed in MDA-MB-231 homogeneous morphology of the hydrophobic core. The
cells. Therefore, the higher cell uptake of PA(D/E)/siRNA hemolysis percentage and cell viability of PADE, PA(D/E), PDE,
resulted in higher gene silence efficiency in MDA-MB-231 cells and P(D/E) micelles were tested and shown in Fig.S2A and B,
than in HepG2 cells. Noticeably, PA(D/E)/siRNA presented very respectively. It was observed PADE micelles showed higher
low cell uptake compared with Lipo2000, but higher or similar hemolysis and lower cell viability than PA(D/E) micelles, and
gene silence were obtained, which was attributed the higher similarly, PDE showed slightly higher hemolysis and lower cell
intracellular endosome escape efficiency than Lipo2000 due to viability than P(D/E) in spite of the P(D/E) with higher positive
suit pKa6.0 certificated in our previous work.29, 39
zeta potential than PDE(Fig.S1B). Beside the possitive charges,
hydrophobic interaction is an important factor to disturb cell
membrane and thus increasing hemolysis and cell toxicity. As
discussion above, the lower zeta potential of PDE micelles than
P(D/E) indicated more D5A moieties with longer hydrophobic
alkyl groups tended to distribute on the core-shell interface of
PDE micelles and led to stronger hydrophobic interaction of PDE
micelles with cell membrane. For P(D/E) micelles, the random
structure of D5A and DEA moieties limited the interface
distribution of D5A moieties, resulting in weaker hydrophobic
interaction bwtween P(D/E) micelles and cells. The effect on
hemolysis and cell toxicity induced by the hydrophobic
interaction outweighed the effect led by slight difference in zeta
potential, so P(D/E) micelles showed higher cell viability
although higher positive zeta potential than PDE micelles. By
the same reason, PA(D/E) showed higher cell viability than PADE
in spite of higher positive zeta potential of the PA(D/E) than
PADE.
Together with the higher cell uptake and siRNA delivery
efficiency, the better cell viability certificated PA(D/E) micelles
could be a kind of potentially biocompatible carrier for siRNA
delivery. At the same time, these results suggested the dilemma
between the higher gene silencing efficiency and lower
biocompatibility for polycationic micelles could be resolved by
the rational structure design of the micelles. Actually, based on
this work, further research works are needed to deeperly
disclose the reasonable polycation structure for balancing the
gene silencing efficiency and biocompatibility.
Intracellular distribution of siRNA-Loaded Micelles
The important way to improve the efficiency of gene silencing
efficiency is to achieve the successful escape of siRNA vector
from the endosome. Confocal laser scanning microscopy (CLSM)
was performed to evaluate the escape capacity and the
intracellular distribution of PADE and PA(D/E)/siRNA complexes
at different N/P ratio. The localization of PADE and
PA(D/E)/siRNA complexes were identified by Cy5 dye labeled
siRNA, Lysotracker Green labeled endosome and DAPI marked
cell nuclei, as shown in Fig. 7A and 7B. The results of the
colocalization ratio of red signals (Cy5-siRNA) in endosomes
were showed in Fig. 7C. Increase of N/P ratios could enhance
the endosomal escape of PADE and PA(D/E)/siRNA complexes,
but the different core morphologies nearly lead to no effect on
the endosomal escape, maybe due to the similar pKa. Our
previous study on PEG blocked PAMA series of amphiphilic
copolymer micelles with different pKa indicated that a narrow
range of pKa 5.8-6.2 facilitated endosomal escape and leaded
to significant enhancement in gene silencing efficiency.39
Therefore, the higher gene silencing efficiency of PA(D/E)/siRNA
than PADE/siRNA was supposed as to the higher internalization.
It could be seen from Fig. 7C, even with the suitable pKa, the
endosomal escape ability of PA(D/E)/siRNA and PADE/siRNA
remained large space to be improved. Definitely, further works
need to be done to explore a more reasonable micellar
structure to enhance the endosomal escape.
In vitro cytotoxicity
Conclusions
The MTT assay was used to evaluate the in vitro cytotoxicity of
PADE and PA(D/E) toward HepG2 cells, and the viability of cells
at 24 h and 48 h were shown in Fig. 8. The increase of siRNA
dose reduced the biocompatibility of siRNA-loaded micelles and
the cytotoxicity of the two copolymer micelles/siRNA was
positively correlated with N/P ratio, owing to the higher charge
density could lead to more damage in cell membrane.45
Significantly, the cell viability of PA(D/E)/siRNA was superior to
PADE/siRNA at various N/P ratios, especially at higher N/P ratio
(N/P= 15) and higher siRNA dose (50nM). Noticeably, the cells
treated with PA(D/E/siRNA (N/P = 5, 10) at 48 hours still kept
high level of viability (Fig. 8C and D).
The dilemma between the higher gene silencing efficiency and
lower biocompatibility keeps egging the design of polycation
micelles as gene vectors. In this work, we obtained the
encouraging results that PA(D/E) with higher positive zeta
potential showed not only better gene silencing efficiency but
In this paper, morphology contribution of the pH-sensitive
hydrophobic core in amphiphipic polycationic micelles to the
siRNA delivery efficiency and biocompatibility was discussed.
Two kinds of amphiphipic poly(aminoethyl methacrylate)
(PAMA) based polycations with different monomer unit
sequences of 2-(diethylamine) ethyl methacrylate (DEA) and 2-
(diamylamine) ethyl methacrylate (D5A) were prepared, i.e.
four-block copolymer mPEG2k-PAMA-PD5A-PDEA (PADE) and
tri-block copolymer mPEG2k-PAMA-P(D5A/DEA) (PA(D/E)). The
PA(D/E) micelle with a homogeneous core due to random
distribution of D5A and DEA units showed higher positive zeta
potential than PADE micelle with a microphase separation
structure. The results obtained from in vitro luciferase assay,
MTT assay and RT-PCR assay indicated that PA(D/E)/siRNA
showed both better cell viability and higher luciferase silencing
efficiency and RRM2 gene knockdown efficiency than
PADE/siRNA. The higher gene silencing efficiency of
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Journal of Materials Chemistry B
Page 6 of 13
ARTICLE
Journal Name
17 M. Gunther, J. Lipka, A. Malek, D. Gutsch, W. Kreyling and A.
PA(D/E)/siRNA was attributed the higher cell uptake, which
indicated higher cell uptake may not result in higher
cytotoxicity. Therefore, the homogenous core structure
together with suit pKa could enhance gene silencing efficiency
and biocompatibility at the same time. That is to say, the
dilemma between the higher gene silencing efficiency and
lower biocompatibility that has troubled polycationic micelles
as gene vectors could be resolved by the rational structure
design of the micellar core.
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Conflicts of interest
There are no conflicts to declare.
25 S. Guo, Y. Huang, W. Zhang, W. Wang, T. Wei, D. Lin, J. Xing, L.
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Acknowledgements
This work was supported by grants from the National Natural
Science Foundation of China (No. 31671021, 81502586, and
81773622).
26 Y. Huang, D. Lin, Q. Jiang, W. Zhang, S. Guo, P. Xiao, S. Zheng,
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45 L. Peng, Y. Gao, Y. N. Xue, S. W. Huang and R. X. Zhuo,
View Article Online
DOI: 10.1039/C8TB02706D
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Table 1. The molecular structure, molecular weight and PDI of PADE and PA(D/E)
Mnb
Polymer name
PADE
molecular structurea
PDIc
3.14
3.22
pKa
6.0
6.0
(104 g/mol)
2.09
mPEG45-PAMA30-PD5A31-PDEA36
mPEG45-PAMA30-P(D5A29-co-DEA29)
PA(D/E)
1.90
aThe molecular structure of the polymer was determined by 1H NMR.
bThe mean molecular weight (Mn) of copolymers was estimated by 1H NMR.
cPDI=Mw/Mn measured by GPC.

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DOI: 10.1039/C8TB02706D
Figure Captions
Scheme 1. Molecular Structures of PADE and PA(D/E) and their self-assemble micelles.
Fig. 1. Synthesis routes of copolymers PADE and PA(D/E).

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Fig. 2. 1H NMR spectra of D5A-MA, PAE, PAED, PAEDE, PAE(D/E), PADE and PA(D/E).
Fig. 3. Characterization of two copolymers: size distribution and TEM images of PADE
(A, C), size distribution and TEM images of PA(D/E) (B, D).

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DOI: 10.1039/C8TB02706D
Fig. 4. (A) The pKa of PADE and PA(D/E). (B) The pH sensitive assembly/disassembly
behavior of PADE. (C) The pH sensitive assembly/disassembly behavior of PA(D/E).
(D)DSC thermograms of PADE and PA(D/E). (E) Zeta potential values of two copolymers
micelles measured by DLS. (F) Agarose gel retardation assay of siRNA-loaded micelles.
Fig. 5. In vitro luciferase expression silencing of PADE and PA(D/E) /siRNA complexes
with the transfection concentrations of siRNA (siRNA: 50 nM) were tested in MDA-MB-
231-Luc cells and HepG2-Luc cells. Among all tests, the cells were treated with different
formulations for 24 h or 48 h. Each bar represents the mean ± SE of three experiments.

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Fig. 6. RT-PCR assay was used to detect the RRM2 gene-silencing efficiencies of
different vectors with the transfection concentrations of 50 nM (A) and 30 nM siRRM2
(B) respectively. The cell uptake detected by flow cytometry in histogram (C) and
quantification(D) in HepG2 cell line. The cell uptake detected by flow cytometry in
histogram (E) and quantification (F) in MDA-MB-231 cell line. The cell uptake was
detected after HepG2 cells and MDA-MB-231 cells were transfected with PADE/siRNA
and PA(D/E)/siRNA complexes for 4h, respectively. Each bar represents the mean ± SE
of six experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 vs naked Cy5-siRNA.

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Fig. 7. Intracellular distribution of siRNA-loaded micelles in HepG2 cells at different N/P
ratios with the transfection concentrations of siRNA were 30 nM (A) and 50 nM (B)
determined by confocal laser scanning microscopy. (C) Statistics of colocated ratios of
Cy5-siRNA and Lysotracker green. Each bar represents the mean ± SE of six
experiments.
Fig. 8. In vitro cell viabilities of PADE and PA(D/E)/siRNA complexes at different N/P
ratios with the transfection concentrations of siRNA were evaluated using MTT assay.
In all tests, the HepG2 cells were treated with different formulations for 24 h or 48 h.
siRNA dose: 30 nM and 50 nM. Each bar represents the mean ± SE of three
experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 vs naked PADE-15.