Cytoplasmic Reactive Cationic Amphiphiles for Efficient Intracellular Delivery and Self-Reporting Smart Release

Article abstract of DOI:10.1021/acs.macromol.5b01110

Nonviral gene delivery vectors need to overcome both extracellular and intracellular obstacles before releasing plasmid DNA in the transcriptionally active form. However, serum and transport stability desired for cationic polymer/pDNA polyplexes contradicts with the eventual plasmid release requirement; chain lengths of cationic polymer vectors render additional compromise between cytotoxicity and transfection efficiency. Although the introduction of stimuli-triggered degradable cationic polymers can partially solve these issues, the quest for novel design criteria and elucidation of elementary cellular transport pathways are highly desirable. Herein we report a supramolecular approach to construct fluorogenic gene delivery vectors via self-assembly of intracellular milieu-reactive cationic amphiphiles. This new type of micellar nanocarriers can effectively bind pDNA to form polyplexes due to multivalent cationic segments at micellar coronas. Upon cellular uptake and endosomal escape, hydrophobic micellar cores are subjected to fluorogenic Michael addition reactions with highly hydrophilic cytoplasmic thiols, leading to micellar disintegration, pDNA release, and emission turn-on for image-guided delivery. (Figure Presented).

Full text of DOI:10.1021/acs.macromol.5b01110

Article
pubs.acs.org/Macromolecules
Cytoplasmic Reactive Cationic Amphiphiles for Efficient Intracellular
Delivery and Self-Reporting Smart Release
Jinming Hu,†,§ Xiao Wang,‡,§ Yinfeng Qian,‡ Yongqiang Yu,*,‡ Yanyan Jiang,† Guoying Zhang,†
and Shiyong Liu*,†
†CAS Key Laboratory of Soft Matter Chemistry, Hefei National Laboratory for Physical Sciences at the Microscale, iChem
(Collaborative Innovation Center of Chemistry for Energy Materials), Department of Polymer Science and Engineering, University
of Science and Technology of China, Hefei 230026, China
‡Department of Radiology, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, China
S
* Supporting Information
ABSTRACT: Nonviral gene delivery vectors need to overcome both extracellular and
intracellular obstacles before releasing plasmid DNA in the transcriptionally active form.
However, serum and transport stability desired for cationic polymer/pDNA polyplexes
contradicts with the eventual plasmid release requirement; chain lengths of cationic
polymer vectors render additional compromise between cytotoxicity and transfection
efficiency. Although the introduction of stimuli-triggered degradable cationic polymers can
partially solve these issues, the quest for novel design criteria and elucidation of elementary
cellular transport pathways are highly desirable. Herein we report a supramolecular
approach to construct fluorogenic gene delivery vectors via self-assembly of intracellular
milieu-reactive cationic amphiphiles. This new type of micellar nanocarriers can effectively
bind pDNA to form polyplexes due to multivalent cationic segments at micellar coronas.
Upon cellular uptake and endosomal escape, hydrophobic micellar cores are subjected to
fluorogenic Michael addition reactions with highly hydrophilic cytoplasmic thiols, leading
to micellar disintegration, pDNA release, and emission turn-on for image-guided delivery.
reductive milieu was utilized to trigger the degradation of
disulfide-containing polycation vectors,37−40 considering the
equilibrium and reversible nature of thiol−disulfide exchange
reactions. In comparison, self-assembling amphiphiles possess-
ing bioresponsive polycation building blocks are more
promising.41−43 Extra polyplexes stability can be envisaged
due to the multivalent pDNA-binding nature, whereas plasmid
release can be actuated via polycation degradation and/or
micelle-unimer equilibrium shift. However, reduction-triggered
cleavage of disulfide linkages are mainly used in the design of
biocleavable polycation amphiphiles, still incurring incomplete
plasmid DNA release;41,42 in addition, micellar polyplexes
possess extended half-lives relative to the duration of the
transfection process. Overall, transfection efficiencies of the
previously described degradable cationic vectors rarely exceed
that of PEI.25−34,37−44 Thus, it is highly desirable to screen
novel design criteria and elucidate elementary cellular transport
pathways. Note that the latter relies on in situ monitoring of
intracellular trafficking and disassembly of polyplexes.45,46
Previously, the abundance of glutathione (GSH) in
cytoplasmic milieu (∼1−10 mM) has been widely exploited
to trigger intracellular payload release from disulfide-containing
nanocarriers and cationic vectors.37−40,47−52 We then speculate
INTRODUCTION
■
Although nonviral gene delivery vectors based on lipids,1
polymers,2−9 and functionalized nanoparticles10−12 have been
developed to alleviate safety issues of recombinant viral vectors,
their delivery efficiency is relatively low.13−17 Specifically,
polyplexes of cationic polymer vectors and plasmid DNA
(pDNA) need to overcome both extracellular and intracellular
obstacles before releasing pDNA for exogenous gene tran-
scription in the cell nucleus. Serum and trafficking stability of
polyplexes is necessary for cationic vectors to prevent pDNA
from unwanted enzymatic degradation. However, this pre-
requisite contradicts with the final crucial step of polyplexes
disintegration and pDNA release. Another dilemma between
transfection efficiency and cytotoxicity also exists for cationic
vectors. Although cationic polymer vectors such as poly(L-
lysine) (PLys), poly(2-(dimethylamino)ethyl methacrylate)
(PDMAEMA), and polyethylenimine (PEI) typically exhibit
enhanced transfection efficiency at higher molecular weights
(MWs), their cytotoxicities are also considerably higher.18−24
These two compromises remarkably limit the design of efficient
nonviral cationic polymer vectors.
In order to solve these major issues, intracellular biodegrad-
able polycations have been designed to promote polyplex
unpackaging and decrease cytotoxicity.25−36 However, polyplex
formation retards polycation degradation kinetics, which might
lead to incomplete plasmid release on the transfection time
scale.32−34 This limitation was getting worse when cytoplasmic
Received: May 22, 2015
Revised: July 9, 2015
Published: August 4, 2015
© 2015 American Chemical Society
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Scheme 1. Supramolecular Aggregates of Thiol-Reactive PyCMA-PDMAEMA Cationic Amphiphiles (P1−P4) as Fluorogenic
a
Intracellular Delivery Nanocarriers
a
Nonfluorescent PyCMA-PDMAEMA amphiphiles (P1−P4) self-assemble into micellar nanoparticles capable of pDNA binding and polyplexes
formation. Upon cellular uptake, endolysosomal escape, and exposure to cytoplasmic reductive milieu, Michael addition reactions of hydrophilic
thiols (e.g., GSH) with PyCMA moieties within hydrophobic cores trigger disintegration of micellar nanoparticles, and the loss of multivalent
interactions then leads to pDNA release; concomitantly, caged PyCMA emission is switched on, allowing for image-guided intracelular pDNA
delivery.
that the integration of GSH-involved irreversible Michael
addition reactions with self-assembling cationic amphiphiles
might offer an excellent gene delivery vector with an
intracellular pDNA “clean” release feature if hydrophilic GSH
addition can render disintegration of cationic micellar
assemblies.53,54 Furthermore, if GSH-relevant Michael addition
is fluorogenic,55,56 we can thus facilely monitor the pDNA
release process. To test the preceding hypothesis, we
synthesized a series of amphiphiles consisting of thiol-reactive
hydrophobic coumarin derivative (i.e., PyCMA) and cationic
PDMAEMA of varying MWs (Scheme 1). Initially, PyCMA-
PDMAEMA spontaneously self-assembles into micelles, ex-
hibiting no coumarin emission due to caging by α,β-
unsaturated ketone in PyCMA. Cationic micellar coronas are
capable of effectively binding pDNA to form polyplexes. Upon
cellular uptake and endosomal escape, cytoplasmic GSH-
mediated addition reactions with hydrophobic PyCMA-
containing cores will lead to micellar disintegration and
pDNA release, the process of which can be in situ monitored
due to emission turn-on of coumarin moieties.
MATERIALS AND METHODS
■
Materials. 2-Dimethylaminoethyl methacrylate (DMAEMA) was
purified by vacuum distillation and stored at −20 °C prior to use. 2,2′-
Azobis(2-methylpropionitrile) (AIBN) was recrystallized from 95%
ethanol. 2-Pyridinecarboxaldehyde, 2-bromoethanol, and Nile red were
purchased from Sigma-Aldrich and used as received. 1,3-Benzenediol,
phosphorus oxychloride (POCl3), ethyl acetoacetate, piperidine, N,N′-
dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP),
glutathione (GSH), and 2-(2-ethoxyethoxy)ethanol (Aladdin) were
purchased from Sinopharm Chemical Reagent Co., Ltd. and used as
received unless otherwise noted. Fetal bovine serum (FBS), penicillin,
streptomycin, and Dulbecco’s modified Eagle’s medium (DMEM)
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Scheme 2. Synthetic Routes Employed for the Preparation of (a) Thiol-Reactive α,β-Unsaturated Ketone-Caged Coumarin
Derivative, (E)-7-(2-Hydroxyethoxy)-3-(3-(pyridin-2-yl)acryloyl)-2H-chromen-2-one (PyCMA−OH, 4) and (b) PyCMA-
Terminated Poly(2- (dimethylamino) ethyl methacrylate) Cationic Amphiphiles, PyCMA-PDMAEMA
were purchased from GIBCO and used as received. Ethidium bromide
(EtBr), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
(MTT), and branched polyethylenimine (Mw = 25 kDa, denoted as
PEI25k) were purchased from Aldrich and used as received. Green
fluorescent protein (GFP)-expressing plasmid DNA (pDNA) and
luciferase-expressing pDNA were purchased from Aldevron and used
as received. Dialysis tube (MWCO = 14 kDa) was purchased from
Shanghai Green Bird Technology Development Co., Ltd. and stored in
1 mM ethylenediaminetetraacetic acid (EDTA) aqueous solution prior
to use. All solvents were of analytical grade and used as received unless
otherwise noted. Water was deionized with a Milli-Q SP reagent water
system (Millipore) to a specific resistivity of 18.4 MΩ·cm.
(44 g, 128 mmol) in acetonitrile (150 mL), dry DMF (12.6 g, 128
mmol), and freshly distilled dry POCl3 (22.6 g, 146 mmol) were added
dropwise with stirring in an ice-water bath. The undissolved salt was
filtered, washed with cold acetonitrile, and recrystallized in water. The
mixture was filtered, washed with cold water, and dried overnight in a
vacuum oven, giving 2,4-dihydroxybenzaldehyde (1) as a white solid
(34 g; yield, 61.6%).
A solution of 1 (10 g, 72.4 mmol), 2-bromoethanol (8.75 g, 70
mmol), and K2CO3 (19.35 g, 140 mmol) in acetone was heated to
reflux at 70 °C. After being stirred overnight under reflux, the reaction
mixture was cooled to room temperature and filtered to remove the
salt. After removing all of the solvents, the residues were dissolved in
CH2Cl2, washed with saline, dried over anhydrous MgSO4, filtered,
and then concentrated on a rotary evaporator. The crude product was
subjected to further purification by silica gel column chromatography
using ethyl acetate and petroleum ether (v/v = 1:2) as the eluent,
affording 2-hydroxy-4-(2- hydroxyethoxy)benzaldehyde (2; 4.2 g;
yield, 32.9%) as a white powder. 1H NMR (CDCl3, ppm, TMS, Figure
Sample Synthesis. Synthetic routes of (E)-7-(2-hydroxyethoxy)-
3-(3-(pyridin-2- yl)acryloyl)-2H-chromen-2-one precursor (PyCMA−
OH, 4) and PyCMA-terminated PDMAEMA amphiphiles (PyCMA-
PDMAEMA, P1−P4) are shown in Scheme 2.
Synthesis of 2-Hydroxy-4-(2-hydroxyethoxy)benzaldehyde (2;
Scheme 2)..57,58 To a well-cooled (0−5 °C) solution of resorcinol
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Table 1. Structural Parameters of PyCMA-Terminated PDMAEMA Cationic Amphiphiles, PyCMA-PDMAEMAn, Used in This
Study
a
b
b
c
d
entry
samples
Mn (kDa)
Mn (kDa)
Mw/Mn
cmc (g/L)
IC50 (μg/mL)
P1
P2
P3
P4
PyCMA-PDMAEMA5
PyCMA-PDMAEMA11
PyCMA-PDMAEMA18
1.1
1.9
3.2
6.8
0.010
0.013
0.035
0.082
71
61
52
2.0
3.0
6.8
1.06
1.09
1.10
PyCMA-PDMAEMA42
a
b
c
1
Calculated from H NMR results. Determined by GPC using THF as the eluent. Determined by surface tensiometry at room temperature.
Determined by cell viability against HepG2 cells via MTT assay.
d
S1a): δ 11.46 (s, 1 H), 9.73 (s, 1 H), 7.44 (d, 1 H), 6.56 (m, 1 H), 6.44
(d, 1 H), 4.14 (m, 2 H), 3.99 (m, 2 H), 2.01 (s, 1H).
According to similar protocols, P2, P3, and P4 were also
synthesized. Their molecular parameters are summarized in Table 1.
Determination of Critical Micellization Concentration. The
critical micellization concentration (cmc) values of P1−P4 were
determined by surface tensiometry. Equilibrium surface tensions were
measured using a JK99B tensiometer with a platinum plate. The
measuring accuracy of the device as reported by the manufacturer is
0.1 mN/m. The reported surface tension values were averaged from
five successive measurements at a temperature of 37.0 0.2 °C. The
cmc values of P1−P4 are summarized in Table 1.
Synthesis of 3-Acetyl-7-(2-hydroxyethoxy)-2H-chromen-2-one (3;
Scheme 2). Ethyl acetoacetate (1.43 g, 11 mmol) and 2 (1.78 g, 9.8
mmol) were dissolved in absolute EtOH (15 mL), and then piperidine
(0.1 g) and AcOH (0.1 mL) were added as catalysts. The mixture was
heated to reflux for 12 h and then was cooled to room temperature to
obtain a bright yellow precipitate. The solid was collected by suction
filtration and washed with cold absolute EtOH. The crude product was
further purified by recrystallization from absolute EtOH, giving 3-
acetyl-7-(2-hydroxyethoxy)-2H-chromen-2-one (3; 1.82 g; yield,
75.3%) as a bright yellow crystal. 1H NMR (CDCl3, ppm, TMS,
Figure S1b): δ 8.49 (s, 1 H), 7.55 (d, 1 H), 6.93 (m, 1 H), 6.85 (d,
1H), 4.19 (t, 1H), 4.04 (t, 1H), 2.71 (s, 3 H), 2.00 (br, 1 H).
Synthesis of (E)-7-(2-Hydroxyethoxy)-3-(3-(pyridin-2-yl)acryloyl)-
2H-chromen-2-one (4; Scheme 2). Compound 3 (1.44 g, 5.8 mmol)
and 2-pyridinecarboxaldehyde (1.24 g, 11.6 mmol) were dissolved in
EtOH/CH3CN (40 mL; v/v = 1:1); piperidine (0.4 g) was then added
as a catalyst. The mixture was heated to reflux for 24 h, and the
solvents were removed under reduced pressure. The crude product
was purified by recrystallization from absolute EtOH, affording (E)-7-
(2-hydroxyethoxy)-3-(3-(pyridin- 2-yl)acryloyl)-2H-chromen-2-one
Reactivity of 4 and P2 toward Thiol-Containing Compounds.
1
The reactivity of PyCMA−OH (4) was investigated by H NMR and
HPLC methods using 2-mercaptoethanol as a model thiol-containing
1
compound (Figure S5). H NMR results suggested the disapperance
of resonance signals characteristic of unsatuated double bond and
HPLC elution profiles exhibited a shorter elution time after treating
with 2-mercaptoethanol, suggesting the complete Michael addition
reaction of 4 with 2-mercaptoethanol (monitored at λ = 365 nm). The
1
reactivity of P2 was also examined by H NMR analysis (Figure S6),
exhibiting a similar reaction propensity as compared to small molecule
4.
Agarose-Gel Retardation Assay. In parallel, the pDNA binding
abilities of P1−P3 before and after treating with GSH (10 mM) were
investigated by agarose-gel retardation assay. The polyplexes solutions
were prepared under a predetermined N/P charge ratio. Electro-
phoresis was carried out on 1% agarose gel with a current of 60 V for
40 min in TAE buffer solution (40 mM Tris-HCl, 1 vol % acetic acid,
and 1 mM EDTA). The retardation of pDNA complexes was
visualized by staining with ethidium bromide. The final pDNA content
was 0.2 μg/well.
1
(4) as a yellow solid (0.83 g; yield, 42.6%). H NMR (CDCl3, ppm,
TMS, Figure S1c): δ 8.72 (d, 1 H), 8.58 (s, 1 H), 8.34 (d, 1 H), 7.85
(d, 1H), 7.76 (m, 1 H), 7.62 (t, 2H), 7.30 (m, 1 H), 6.97 (m, 2 H),
4.23 (t, 2 H), 4.07 (t, 2H), 2.00 (br, 1 H). HRMS (m/z): calcd for
C19H15NO5, 337.10; found, 338.10184 [M + H]+ (Figure S2).
Synthesis of PyCMA-PDMAEMAn (Scheme 2). Typical procedures
employed for the RAFT synthesis of cationic PDMAEMA
homopolymers are as follows.59 Using the preparation of PDMAEMA5
as an example, into a reaction tube equipped with a magnetic stirring
b a r , D M A E M A ( 2 . 0 g , 1 2 . 7 m m o l ) , 4 - c y a n o - 4 -
(phenylcarbonothioylthio)pentanoic acid (586 mg, 2.1 mmol), AIBN
(49 mg, 0.3 mmol), and 1,4-dioxane (4 mL) were charged. The
reaction tube was carefully degassed by three freeze−pump−thaw
cycles and then sealed under vacuum. After thermostatting at 70 °C in
an oil bath and stirring for 6 h, the reaction tube was quenched into
liquid nitrogen and opened and the reaction mixture was diluted with
THF and then precipitated into an excess of petroleum ether. The
preceding dissolution−precipitation cycle was repeated three times.
PDMAEMA5 was obtained as reddish liquid (1.54 g; yield, 59.6%).
PyCMA-PDMAEMA5 (P1) was prepared by the esterification of
PDMAEMA5 with PyCMA−OH (4) in the presence of DCC and
DMAP. In a typical procedure, PDMAEMA5 (100 mg, 0.1 mmol) was
dissolved in anhydrous toluene (10 mL), and azeotropic distillation
was carried out at 50 °C under reduced pressure to remove most of
the solvent. Compound 4 (40 mg, 0.12 mmol) and dry CH2Cl2 (10
mL) were added. After cooling to 0 °C in an ice-water bath, a CH2Cl2
solution (10 mL) containing DCC (24 mg, 0.12 mmol) and DMAP
(2.4 mg, 0.02 mmol) mixture was added dropwise over 1 h. The
reaction mixture was stirred at room temperature for 24 h. After
removing insoluble salt by filtration, the filtrate was concentrated on a
rotary evaporator and then precipitated into an excess of cold diethyl
ether. The preceding dissolution−precipitation cycle was repeated
three times. After drying in a vacuum oven overnight at room
temperature, PyCMA-PDMAEMA5 (P1) was obtained as a yellowish
powder (80 mg; yield, 57%).
Ethidium Bromide Replacement Assay..34,43 Compound P2
micellar solution (0.2 g/L) in 10 mM PBS buffer (pH 7.4) was
mixed with pDNA to form polyplexes at an N/P ratio of 10. The
polyplexes were mixed with EtBr (final concentration, 0.1 mg/mL)
and incubated at room temperature for 15 min. After that, the ternary
solution was divided into two aliquots. One aliquot was treated with
GSH (final concentration, 10 mM). Subsequently, fluorescence spectra
of the mixture solutions were recorded on a fluorescence spectrometer
with the excitation and emission wavelengths at 510 and 590 nm,
respectively.
Cellular Uptake Process of PyCMA-PDMAEMA11 (P2) Micelles
Loaded with Nile Red Observed by Confocal Laser Scanning
Microscopy (CLSM). HepG2 cells were plated onto Petri dishes (In
Vitro Scientific, 35 mm dish with 20 mm bottom well) at a density of 1
× 106 cells per dish and cultured in Dulbecco’s modified Eagle
medium (DMEM) supplement with 10% fetal bovine serum (FBS),
penicillin (100 units per mL), and streptomycin (100 μg mL−1) for 24
h at 37 °C in CO2/air (5:95). Nile-red-loaded P2 micellar solution was
added to the Petri dishes at a final concentration of 0.05 g/L. After
being incubated for 1 and 8 h, the cells were washed with PBS for
three times. The fluorescence images were then taken on the
microscope. The blue fluorescence from activated PyCMA moieties
was observed using a 405 nm laser with the emission channel set to be
420−470 nm. Fluorescence of Nile red was observed using a 592 nm
laser with the emission channel set to be 620−700 nm. Late
endosomes and lysosomes of the cells were stained with LysoTracker
Green (200 nM) after 30 min of incubation before imaging and
observed using a 488 nm laser, and the emission wavelength was read
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Figure 1. (a, b) TEM images and (c, d) AFM images recorded for (a, c) P2 micelles and (b, d) P2/pDNA polyplexes at an N/P ratio of 10. AFM
images of P2/pDNA polyplexes upon treating with 10 mM GSH for (e) 30 and (f) 200 min. (g) Hydrodynamic diameter distributions, f(Dh),
recorded for micellar solutions (0.2 g/L) of P1, P2, P1/pDNA, and P2/pDNA polyplexes at an N/P ratio of 10, respectively. (h) ζ potential
recorded for P2/pDNA polyplexes at varying N/P ratios.
from 515 to 560 nm and expressed as green. Cellular uptake of P2
micelles and P2/Cy5-labeled pDNA polyplexes were observed
similarly. The intracellular distribution of P2/Cy5-labeled pDNA
polyplexes was quantitatively calculated by the co-localization ratio of
blue/green and red/green fluorescence pixels with LysoTracker Green
pixels.
determination of luciferase-expressing pDNA gene transfection, the
medium was removed and the cells were rinsed with PBS. The cells in
each well were treated with 200 μL of cell lysis buffer followed by
freeze−thaw cycles three times to ensure complete lysis. The cell lysate
was transferred into a 200 μL vial and centrifuged for 5 min at 12,000
rpm, and the supernatant was collected for luminescence measure-
ments. Luciferase gene expression was evaluated using the Luciferase
Assay System (Promega Co., Madison, WI, USA). Bioluminescence
signals were measured using a 96-well microplate luminometer with an
exposure time of 10 s. The amount of total cellular protein was
determined by the BCA protein assay kit (Pierce, Waltham, MA,
USA). The final results were reported in terms of relative light units
(RLU) per milligram of protein. PEI25k was employed as a positive
control at an N/P ratio of 3.
In Vitro Cytotoxicity Assay. HepG2 cell line was employed to
evaluate the in vitro cytotoxicity of polymers via a MTT assay. HepG2
cells were first cultured in Dulbecco’s modified Eagle medium
(DMEM) supplemented with 10% fetal bovine’ serum (FBS),
penicillin (100 units/mL), and streptomycin (100 μg/mL) at 37 °C
in a CO2/air (5:95) incubator for 2 days. For cytotoxicity assay,
HepG2 cells were seeded into 96-well plates at a density of 5000 cells/
well. Afterward, the cells were incubated for 24 h, then DMEM was
replaced with fresh media, and the cells were treated with polymer
solutions with varying concentrations; the treated cells were incubated
in a humidified environment with 5% CO2 at 37 °C for 48 h. The
MTT reagent (in 20 μL of PBS, 5 mg/mL) was added to each well.
The cells were further incubated for 4 h at 37 °C. The medium in each
well was then removed and replaced by 180 μL of DMSO. The plate
was gently agitated for 15 min before the absorbance at 570 nm was
recorded by a microplate reader (Thermo Fisher). Each experiment
condition was done in quadruplicate, and the data are shown as the
mean value plus a standard deviation ( SD).
In Vitro Transfection and Gene Expression. HepG2 cells were
seeded at a density of 5 × 103 cells/well in DMEM media
supplemented with 10% FBS in 96-well plates and cultured for 24 h.
Before adding polyplexes, the serum-containing culture medium was
replaced with 100 μL of serum-free medium. The polyplexes were then
added to reach a content of 200 ng of DNA/well at an N/P ratio of 10
and the mixture was incubated for 4 h at 37 °C. The culture medium
was then replaced with 200 μL of fresh complete medium, and the
cells were incubated for an additional 48 h. Transfection experiments
were performed in triplicate. After incubation, the medium was
removed and the cells were rinsed once with PBS. For the
determination of GFP-expressing pDNA gene transfection, the GFP-
positive cells were observed using fluorescence microscopy. For the
Characterization. All nuclear magnetic resonance (NMR) spectra
were recorded on a Bruker AV300 NMR spectrometer (resonance
1
frequency of 300 MHz for H) operated in the Fourier transform
mode. CDCl3 was used as the solvent. Molecular weights and
molecular weight distributions were determined by GPC using a series
of three linear Styragel columns HR2, HR4, and HR5 and an oven
temperature of 45 °C. A Waters 1515 pump and a Waters 2414
differential refractive index detector (set at 30 °C) were used. The
eluent was THF at a flow rate of 1.0 mL min−1. A series of six
polystyrene standards with molecular weights ranging from 800 to
400000 g mol−1 were used for calibration. Dynamic light scattering
(DLS) measurements were conducted on a Malvern Zetasizer Nano
ZS. All data were averaged over three consecutive measurements. All
samples were filtered through 0.45 μm Millipore Acrodisc-12 filters to
remove the dust. Transmission electron microscopy (TEM)
observations were conducted on a Hitachi H-800 electron microscope
at an acceleration voltage of 200 kV. The samples for TEM
observations were prepared by placing 10 μL of the micellar solution
(0.5 g L−1) on copper grids coated with thin films of Formvar and
carbon successively. AFM measurements were performed on a Digital
Instrument Multimode Nanoscope IIID operating in the tapping
mode under ambient conditions. Silicon cantilever (RFESP) with
resonance frequency of ∼80 kHz and spring constant of ∼3 N/m was
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Figure 2. (a) Evolution of scattered light intensity of P2 micellar solution (0.2 g/L) upon addition of 10 mM GSH at pH 7.4. (b) Hydrodynamic
diameter distributions, f(Dh), recorded for P2 micelles before and after treating with GSH for 60 min. (c) Time-dependent evolution of OD600
during formation and dissociation (adding GSH) of P2/pDNA polyplexes at N/P ratio of 10. Evolution of (d) fluorescence emission spectra (λex
=
370 nm) and (e) emission intensity changes at 416 nm recorded for P2/pDNA polyplexes upon treating with GSH at pH 7.4. (f) Macroscopic
images recorded for P2/pDNA polyplexes before and after treating with GSH under ambient light (left) and UV irradiation (right).
used. The set-point amplitude ratio was maintained at 0.7 to minimize
sample deformation induced by the tip. The samples were prepared by
dip coating 0.05 g/L aqueous micellar solutions onto the surface of
freshly cleaved mica. Confocal laser scanning microscopy (CLSM)
longer cationic blocks render higher cmc values. Taking
images were acquired using a Leica TCS SP5 microscope. HPLC
terminal moiety and hydrophilic PDMAEMA segment, P1−P4
exhibit amphiphilic features with critical micellization concen-
trations (cmcs) in the range of 10−80 mg/L (Table 1), and
PyCMA-PDMAEMA11 (P2) as an example, it self-assembles
into spherical micelles in aqueous media, as evidenced by both
TEM and AFM observations (Figure 1a,c).
analysis was performed with a Shimadzu HPLC system, equipped with
a LC-20AP binary pump, a SPD-20A UV−vis detector, and a
Symmetry C18 column. All UV−vis spectra were acquired on a TU-
1910 double-beam UV−vis spectrophotometer (Puxi General
Instrumental Co., Beijing, China). Fluorescence spectra were recorded
on an F-4600 (Hitachi) spectrofluorometer. The slit widths were set at
5 nm for both excitation and emission.
Possessing a pKa of ∼7.3, PDMAEMA homopolymers with
high MWs have been utilized as gene delivery vectors.20−23,60,61
Although P2 unimers are of low MW (<2 kDa) and unable to
effectively condense pDNA,21−23 P2 micellar nanoparticles can
condense GFP-expressing pDNA due to the multivalent nature
of pDNA-binding micellar coronas (Scheme 1).62−65 Upon
pDNA introduction, the size of P2 micellar aggregates
increased considerably due to formation of micellar polyplexes
(Figure 1b,d). Dynamic light scattering (DLS) revealed the
increase of average hydrodynamic diameter, ⟨Dh⟩, from ∼110
to ∼360 nm (Figure 1g). Moreover, ζ potential values evolved
from −32 to +18 mV in the N/P range of 0−20 (Figure 1h).
RESULTS AND DISCUSSION
■
Hydroxyl-terminated and α,β-unsaturated ketone caged cou-
marin derivative (PyCMA−OH, 4) was synthesized at first.
Carboxyl-terminated PDMAEMA, synthesized by a controlled
radical polymerization and subsequent end group esterification
reaction with 4, afforded PyCMA-PDMAEMAn of different
cationic block lengths (P1−P4; Scheme 2). All intermediate
precursors and final products were characterized in detail
(Figures S1−S4). Table 1 summarizes structural parameters of
P1−P4. Due to the presence of both hydrophobic PyCMA
1
For both 4 and P2, HPLC and H NMR analysis verified the
efficient generation of thiol adducts through Michael addition
reaction with 2-mercaptoethanol, as evidenced from the
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disappearance of unsaturated double bond signals (Figures S5
and S6). Thus, the reactivity of the PyCMA moiety was
retained when installed at the chain terminal of cationic
amphiphiles.54 We then postulated that as a thiol-containing
highly hydrophilic tripeptide, Michael addition reaction of
cytosol-rich GSH with P1−P4 aggregates should lead to
micellar disintegration if hydrophobic PyCMA can be rendered
hydrophilic (Scheme 1).
Next, we monitored the evolution of UV−vis absorbance
spectra of P2 aqueous solution upon GSH addition. During co-
incubation, a slight absorbance decrease at ∼290 nm and
hypochromatic peak shift from ∼370 to ∼360 nm were
discerned (Figure S7a). The steady decrease of absorbance
baseline at ∼700 nm was also clearly evident, suggesting the
disassembly of P2 micelles upon GSH treating (Figure S7b).
Micellar disintegration was further confirmed by the prominent
decrease of scattering light intensities and ⟨Dh⟩ (Figures 2a,b).
Both Michael addition reaction and micellar disassembly
processes were completed ∼30 min after GSH addition at
pH 7.4 (Figure 2a). For P1, P3, and P4 micelles, GSH-
triggered micellar disintegration was achieved in all cases
(Figure S8).
Figure 3. Agarose-gel electrophoresis of P1−P3/pDNA polyplexes at
various N/P ratios before (left) and after (right) treating with 10 mM
GSH.
To clarify whether GSH addition could elicit unpackaging of
P2/pDNA micellar polyplexes as well, the change in optical
density (OD600) of P2 micellar solution was monitored. Prior
to pDNA addition, the OD600 was lower than 0.02, which is
consistent with the formation of small aggregates (Figure 1).
When pDNA was introduced, the OD600 remarkably increased
to 0.17 within ∼50 min (Figure 2c), which corresponds to the
polyplex formation process. However, upon addition of 10 mM
GSH, an abrupt decrease in OD600 value relevant to the
disassembly of polyplexes was observed (Figure 2c). GSH-
triggered disassembly of polyplexes was also manifested by
AFM results, and intermediate pDNA condensing nanostruc-
tures and free pDNA loops can be discerned after 30 and 200
min co-incubation with GSH, respectively (Figures 1e,f).
Moreover, fluorescence emission turn-on along with polyplex
disintegration was achieved, exhibiting an overall ∼28.5-fold
increase (Figure 2d,e). Concurrent polyplex disassembly and
emission turn-on can also be visually checked (Figure 2f).
Taken together, Michael addition reaction of PyCMA moieties
with GSH-actuated disintegration of both PyCMA-PDMAEMA
micelles and micellar pDNA polyplexes; due to the fluorogenic
nature of PyCMA-GSH addition reaction, this process also
leads to emission turn-on, which can serve as a self-reporting
delivery module.
Agarose-gel retardation assay was further employed to verify
GSH-triggered pDNA release from micellar polyplexes (Figure
3). Before GSH treating, the pDNA retardation ability of P1−
P3 gradually increased with elevated N/P ratios, and cationic
amphiphiles with higher MW exhibited improved retardation at
fixed N/P ratios.21−23 Specifically, P1 and P2 micelles exhibited
complete retardation at N/P ratios of 8 and 3, respectively.
These results indicated the formation of nonstoichiometric
complexes, which are in dynamic equilibrium with possibly
present free unimers/micelles, upon mixing pDNA and an
excess amount of cationic vectors. To the best of our
knowledge, previously reported standard protocols for the
fabrication of polyplexes from pDNA and cationic vectors do
not involve the purification step.2−9 However, we firmly believe
that the elucidation of this issue is crucial for understanding the
polymer physics behind polyplexes formation. In subsequent
sections, these polyplexes with net positive surface charges were
directly utilized without further purification.
However, after GSH treatment, P1 can no longer efficiently
condense pDNA even at an N/P ratio of 20; whereas for P2,
complete retardation was achieved unless the N/P ratio was
higher than 15. Notably, GSH treatment exerted negligible
effect on the retardation ability of P3, although DLS
measurements revealed reorganization of P3 micellar poly-
plexes upon GSH treating (Figure S8c,d). We therefore
inferred that P3(GSH) adducts can still interact with pDNA
and form structurally loose polyplexes at comparable N/P
ratios. Ethidium bromide (EtBr) replacement assay was further
explored to evaluate GSH-actuated pDNA release. Upon
incubating P2/pDNA polyplexes with EtBr probe in the
presence of 10 mM GSH at pH 7.4, a steady emission increase
originating from EtBr intercalation into free pDNA strands can
be discerned upon extended incubation (Figure S9).34,43 In
sharp contrast, negligible emission increase was detected
without GSH addition. These results further confirmed
polyplexes disassembly and spontaneous pDNA release
triggered by GSH.
Upon cellular uptake, the endocytic transport of polyplexes is
typically associated with early/late endosomes and lysosomes
before entering into cytosol. pDNA release in these acidic
organelles need to be avoided due to unwanted enzymatic
degradation. Michael addition reaction of P2/pDNA polyplexes
with GSH was then checked at ∼ pH 5.5, mimicking
intracellular acidic organelles. Almost no emission increase
was discerned upon co-incubation, confirming that Michael
addition reaction between GSH and PyCMA moieties is
negligible under acidic conditions (Figure S10).66 This feature
is quite advantageous for PyCMA-PDMAEMA micelles as
delivery vectors because premature pDNA release in
endolysosomes can be prohibited and “clean” plasmid release
can be solely and safely actuated in the reductive and neutral
cytosol milieu.
Encouraged by the preceding results, we further investigated
cellular gene delivery performance of PyCMA-PDMAEMA
amphiphiles. Upon incubation with P2 micelles for 24 h,
initially nonfluorescent P2 micelles showed intense blue
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Figure 4. (a) CLSM images of HepG2 cells after co-incubation with P2/Cy5-pDNA polyplexes at an N/P ratio of 10. Late endosomes and
lysosomes were stained with LysoTracker Green. Co-localization ratio analysis (b) between blue channel emission from activated PyCMA and green
channel emission of stained endolysosomes and (c) between red channel emission from Cy5-pDNA and green channel emission of stained
endolysosomes.
emission outside endolysosomes, suggesting that P2 micelles
can be efficiently taken up by HepG2 cells, followed by
endosomal escape into cytosol and emission turn-on by virtue
of cytosolic GSH-aided Michael addition reaction (Figure S11).
To check whether cytosolic GSH-mediated addition reaction
could disintegrate P2 micelles, a polarity-sensitive dye, Nile red,
was encapsulated and intracellular emission evolution was
monitored (Figure S12). After 1 h incubation, negligible blue
emission and intense red emission was observed, and good co-
localization between LysoTracker green and Nile red emissions
revealed that P2 nanoparticles were located in acidic organelles.
This should be ascribed to retarded Michael addition reactivity
within acidic endolysosomes (Figure S10). Upon extending the
incubation time to 8 h, intense blue emission and diminished
red emission were observed. This confirmed endosomal escape
of P2 micelles from acidic organelles and disassembly of P2
micelles triggered by GSH addition in the neutral cytosolic
milieu since Nile red probe is almost nonfluorescent in the
hydrophilic milieu.
located in acidic organelles. Upon extended incubation, blue
emission from GSH-activated PyCMA steadily increased
(Figure 4a). The co-localization ratio between coumarin blue
and LysoTracker green channels always remained less than
20%, irrespective of incubation duration (Figure 4b). Addi-
tionally, the co-localization ratio between green channel and
Cy5-pDNA red channel prominently decreased from 83.5% at
1 h incubation to 22.3% at 24 h incubation (Figure 4c),
suggesting considerable endosomal escape of polyplexes into
the cytosol. Most importantly, the discrete distribution of blue
and red emissions confirmed the disassembly of polyplexes and
pDNA release.
To further verify that cytoplasmic GSH is responsible for
intracellular disassembly of P2/pDNA polyplexes, hydrophobic
1-propanethiol (PPT) was utilized as a control. Upon addition
of PPT into P2 micellar solution, no distinct PyCMA emission
increase could be detected despite that UV−vis spectra revealed
that Michael addition reactions did occur (Figure S13);
moreover, neither scattered intensity nor ⟨Dh⟩ exhibited
appreciable changes. All of these results revealed no
disintegration occurred for P2 micelles. The negligible emission
increase of PyCMA moieties after PPT addition should be
ascribed to the self-quenching effect of activated coumarin dyes
within hydrophobic micellar cores (Figure S13b). Accordingly,
PPT treating also cannot trigger pDNA release from P2/pDNA
Subsequently, Cy5-labeled pDNA was employed to elucidate
intracellular disassembly of micellar polyplexes (Figure 4). After
incubating HepG2 cells with polyplexes for 1 h, no appreciable
blue emission from activated PyCMA was detected. The
overlap between LysoTracker green and Cy5 red channel
emissions suggested that P2/Cy5-pDNA polyplexes were
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micellar polyplexes (Figure S14). Cell viability assays revealed
that P3, with a longer cationic block, exhibited higher
cytotoxicity (Figure S15a).19,20 The IC50 of P2 was determined
to be ∼61 μg/mL (Table 1), which is much lower than that of
PEI25k (IC50 ∼ 9 μg/mL).19−23 Moreover, the cytotoxicity of
P2 could be further decreased after GSH treating (Figure
S15b). In contrast, increased cytotoxicity of P2 micelles after
PPT treating was observed due to that they cannot be easily
dissociated. In a further step, in vitro cell transfection
experiments were conducted on HepG2 cells by using GFP-
expressing pDNA. After GFP pDNA transfection using P2
micelles as a cationic vector at an N/P ratio of 10, both blue
emission from activated PyCMA and green emission from GFP
can be detected after 48 h incubation (Figure S16a). In the
control experiment, preincubation of P1 and P2 amphiphiles
with GSH significantly decreased transfection efficiencies
(Figure S17), implying the importance of multivalent pDNA
binding due to self-assembly of cationic amphiphiles (Scheme
1).
Finally, the transfection efficiency was quantified using
luciferase-expressing pDNA as the reporter gene. Before
treating with thiol-containing compounds and at a fixed N/P
ratio of 10, P2 displayed optimal gene transfection efficiency,
which was ∼30% relative to that of PEI25K at an N/P ratio of 3
(Figure S16b). Note that previous reports revealed that
PDMAEMA with an MW of ∼15 kDa was not effective and
a minimum MW of ∼43 kDa was required for efficient gene
transfection.20 We thus established for the first time that
PDMAEMA bearing a GSH-reactive hydrophobic terminal
moiety with much lower MW (<2 kDa) can serve as efficient
gene delivery vectors through supramolecular engineering; note
that PyCMA-PDMAEMA cationic amphiphiles also possess
extra advantages including lower cytotoxicity (Figure S15) and
image-guided pDNA release (Figures 3 and 4). After GSH
treating, P1 and P2 micelles were prone to disassembly,
exhibiting prominently compromised gene transfection effi-
ciency. Moreover, upon preincubation with PPT, elevated
cytotoxicity might account for decreased transfection efficien-
cies (Figure S16b).
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.macro-
mol.5b01110.
■
S
Additional characterization data including NMR spec-
troscopy, GPC results, UV−vis spectra, DLS, fluores-
cence spectra, confocal images, and MTT assay (PDF)
AUTHOR INFORMATION
Corresponding Authors
*E-mail: sliu@ustc.edu.cn (S.L.).
*E-mail: hfyuyongqiang@hotmail.com (Y.Y.).
■
Author Contributions
§J.H. and X.W. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The financial support from the National Natural Scientific
Foundation of China (NNSFC) project (Grant Nos. 21274137,
51273190, 51033005, and 51473153), the Fundamental
Research Funds for the Central Universities, and the
Specialized Research Fund for the Doctoral Program of Higher
Education (SRFDP, Grant 20123402130010) is gratefully
acknowledged.
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