Neuron-targeted copolymers with sheddable shielding blocks synthesized using a reducible, RAFT-ATRP double-head agent

Article abstract of DOI:10.1021/ja3085803

Adaptation of in vitro optimized polymeric gene delivery systems for in vivo use remains a significant challenge. Most in vivo applications require particles that are sterically stabilized, which significantly compromises transfection efficiency of materi

Full text of DOI:10.1021/ja3085803

Communication
pubs.acs.org/JACS
Neuron-Targeted Copolymers with Sheddable Shielding Blocks
Synthesized Using a Reducible, RAFT-ATRP Double-Head Agent
Hua Wei, Joan G. Schellinger, David S. H. Chu, and Suzie H. Pun*
Department of Bioengineering and Molecular Engineering and Sciences Institute, University of Washington, Seattle, Washington
98195, United States
S
* Supporting Information
Scheme 1. Polymer Structure, DNA Condensation, Cell
Binding, Endocytosis, and Proposed Route for Subsequent
Reduction-Triggered Intracellular Gene Release
ABSTRACT: Adaptation of in vitro optimized polymeric
gene delivery systems for in vivo use remains a significant
challenge. Most in vivo applications require particles that
are sterically stabilized, which significantly compromises
transfection efficiency of materials shown to be effective in
vitro. We present a multifunctional well-defined block
copolymer that forms particles useful for cell targeting,
reversible shielding, endosomal release, and DNA
condensation. We show that targeted and stabilized
particles retain transfection efficiencies comparable to the
nonstabilized formulations. A novel, double-head agent
that combines a reversible addition−fragmentation chain
transfer agent and an atom transfer radical polymerization
initiator through a disulfide linkage is used to synthesize a
well-defined cationic block copolymer containing a
hydrophilic oligoethyleneglycol and a tetraethylenepent-
amine-grafted polycation. This material effectively con-
denses plasmid DNA into salt-stable particles that deshield
under intracellular reducing conditions. In vitro trans-
fection studies show that the reversibly shielded polyplexes
afford up to 10-fold higher transfection efficiencies than
the analogous stably shielded polymer in four different
mammalian cell lines. To compensate for reduced cell
uptake caused by the hydrophilic particle shell, a neuron-
targeting peptide is further conjugated to the terminus of
the block copolymer. Transfection of neuron-like, differ-
entiated PC-12 cells demonstrates that combining both
targeting and deshielding in stabilized particles yields
formulations that are suitable for in vivo delivery without
compromising in vitro transfection efficiency and are thus
promising carriers for in vivo gene delivery applications.
when used in vivo, they must preferentially transfect the target
cell type rather than the vast majority of other cells present.
To address the aggregation issue, a hydrophilic polymer shell,
e.g. poly(ethylene glycol) (PEG) or N-(2-hydroxypropyl)-
methacrylamide (HPMA), is incorporated into most polyplexes
designed for in vivo use.4 However, polyplexes shielded against
protein adsorption and aggregation are also poorly recognized
and internalized by cells, compromising transfection efficiencies.
Formulations with reversible deshielding properties that are
triggered by acid conditions (found in tumor microenvironments
or in endosomes after cellular uptake) or reducing conditions
(found in the cell cytosol) are generally more efficient than
formulations with stable polymer shields. Notable examples have
been tested for in vivo delivery: Wagner’s ternary complexes
containing targeting ligand conjugated to one polycation, PEG
conjugated to a second polycation via an acid-labile hydrazone,
and plasmids; Wang’s ternary complexes of polyplexes coated
with a charge-reversing polymer that deshield in mildly acidic
environments; and Kataoka’s block copolymers of PEG and
poly[Asp(DET)] that deshield in reducing conditions.5
However, to our knowledge, the formulations reported to date
olycations are attractive for gene delivery because they self-
Passemble with and condense nucleic acids, can be
synthesized at large scale, and offer flexible chemistries for
functionalization.1 Many polymer compositions and architec-
tures have been synthesized, and in vitro screening of these
polymers has yielded many materials that efficiently transfect
2
cultured mammalian cells, but only a small subset of these
materials is suitable for in vivo use due to additional extracellular
barriers. Polyplexescomplexes of polycations and nucleic
acidsare colloids typically unstable in physiological con-
ditions.3 They are prone to protein adsorption and aggregation,
which can lead to inflammation and mortality. Furthermore,
Received: August 29, 2012
Published: September 26, 2012
© 2012 American Chemical Society
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Journal of the American Chemical Society
Communication
do not incorporate targeting ability and/or require multiple
polymer components in the final structures, complicating scale-
up and manufacturing.
P(GMA-TEPA), by a combination of RAFT polymerization of
oligo(ethylene glycol) monomethyl ether methacrylate
(OEGMA) and ATRP of glycidyl methacrylate (GMA), followed
by postpolymerization decoration of reactive epoxy groups in the
P(GMA) block by tetraethylenepentamine (TEPA) (Figure 1b).
Diblock copolymers synthesized using this double-head agent
possess several notable qualities: (1) They have well-controlled
composition with narrowly distributed molecular weight. (2)
They can be easily modified with a targeting ligand at the outer
corona. (3) The “in vivo ready” diblock formulation shows
transfection efficiency similar to that of the in vitro optimized
polycation segment due to the reversible shielding combined
with targeting ability. The neuron targeting peptide Tet1-
conjugated P(OEGMA)15-SS-P(GMA-TEPA)50 is expected to
transfect cells by condensing DNA efficiently to form core−shell
type polyplexes with the P(GMA-TEPA)/DNA electrostatic
complex as the core and the P(OEGMA) block as the shell. Once
internalized, the polyplexes become localized within the
endocytic vesicles. The protonatable amines in TEPA were
included to facilitate endosomal escape through the proton
sponge effect,6 and glutathiones in the intracellular environment
are expected to degrade the disulfide links, leading to detachment
of the hydrophilic P(OEGMA) coating and release of DNA
(Scheme 1).
The major advancement reported in this work is the
development of a polymeric nucleic acid carrier that incorporates
cell targeting, reversible colloidal stability, and efficient intra-
cellular delivery into a single well-defined material (Scheme 1).
The key to this is a novel, reducible double-head agent consisting
of both a reversible addition−fragmentation chain transfer
(RAFT) agent and an atom transfer radical polymerization
(ATRP) initiator connected by a disulfide bond (Figure 1a).
We used this double-head agent to synthesize an optimized
reduction-responsive cationic block copolymer, P(OEGMA)-SS-
The double-head agent CPADB-SS-iBuBr was synthesized by
N,N′-dicyclohexylcarbodiimide coupling between the RAFT
chain transfer agent (CTA) 4-cyanopentanoic acid dithioben-
zoate (CPADB)7 and 2-hydroxyethyl-2′-(bromoisobutyryl)ethyl
disulfide initiator (OH-SS-iBuBr)8 in the presence of 4-
(dimethylamino)pyridine as a catalyst. After purification by
column chromatography, CPADB-SS-iBuBr was successfully
obtained with 43.1% yield (Figure 1a).
The most commonly used hydrophilic shell employed in
polyplex stabilization is PEG, which offers facile incorporation by
various methods, e.g. using a PEG-based macroinitiator,5a,9
macro-CTA,10 or a coupling reaction.11 Coupling through
disulfide-thiol exchange usually requires synthesis of both a
mercapto-functionalized and a pyridyl disulfide-functionalized
polymer;11 thus it involves reaction between large macro-
molecules and extra separation steps to remove the excess
homopolymers from desired block copolymers. In addition to
PEG, OEGMA and HPMA are biocompatible, hydrophilic, shell-
building units of nanocarriers.12,13 However, there are no
published reports thus far on the synthesis of well-defined
copolymers containing sheddable OEGMA and HPMA blocks
by controlled living radical polymerization (CLRP). Chain
extension using different monomers by consecutive CLRP
processes always leads to block copolymers with nondegradable
C−C links in the block junctions. Oh et al. reported the synthesis
of poly(lactide)-SS-polymethacrylate amphiphilic block copoly-
mers with SS linkages positioned at the block junction by ring-
opening polymerization and ATRP,8 but these methods are
limited to cyclic monomers.
The CPADB-SS-iBuBr double-head agent developed herein
offers a simple and versatile means to prepare block copolymers
based on diverse hydrophilic and hydrophobic monomers with
cleavable links in the block junctions for various applications.
The design principle of the double-head agent integrates RAFT
and ATRP techniques, taking advantage of their different
mechanisms for orthogonal synthetic strategy. The resulting
double-head agent was first used as a RAFT CTA to polymerize
both OEGMA (Mn ≈ 300) and HPMA. The RAFT kinetics of
OEGMA (Table S1) and HPMA (Table S2) polymerization
Figure 1. (a) 1H NMR spectrum of the double-head agent (CPADB-SS-
iBuBr). (b) Synthesis of Tet1-P(OEGMA)15-SS-P(GMA-TEPA)50.
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Communication
using CPADB-SS-iBuBr was monitored by 1H NMR and SEC-
MALLS. Both monomers were polymerized with first-order
kinetics (Figure S2a−c) and low PDI (<1.3), demonstrating
excellent synthesis control via RAFT polymerization.
The hydrophilic OEGMA and HPMA blocks were then used
as macroinitiators for ATRP of GMA. To minimize the
possibility of concurrent RAFT-ATRP,14 GMA, which is
polymerized with fast kinetics by ATRP but with slower kinetics
by RAFT, was polymerized with short reaction time. We
demonstrate by NMR and gel permeation chromatography that
polymerization of GMA occurs predominantly by ATRP (see
Supporting Information (SI)). Block copolymers of P-
1
(OEGMA)-SS-P(GMA) were characterized by H NMR to
verify successful polymerization and to determine the degree of
polymerization (DP) (Figure S1b). P(GMA) contains pendant
reactive epoxy groups that were further functionalized by TEPA
to generate the polycation block. The final diblock copolymer
1
was characterized by H NMR (Figure S1c) and SEC-MALLS
(Figure S2d). In earlier work we optimized the polycation block,
screening various oligoamine and lengths of polymer backbone
for optimal cell transfection efficiency (data not shown). From
this initial screen, we identified P(GMA) of DP 50 grafted with
TEPA, P(GMA-TEPA)50 (Mn = 22.5 kDa, PDI = 1.11, dn/dc =
0.216), to be the most effective carrier. Its transfection
efficiencies were comparable to that of branched poly-
(ethylenimine) (bPEI, 25 kDa). We further tested diblocks of
P(GMA-TEPA)50 with P(OEGMA) and P(HPMA) of various
lengths and identified an optimal material, P(OEGMA)15-SS-
P(GMA-TEPA)50 (Mn = 31.5 kDa, PDI = 1.29, dn/dc = 0.202).
As a control, the reduction-insensitive P(OEGMA)15-b-P(GMA-
TEPA)50 copolymer (Mn = 30.1 kDa, PDI = 1.28, dn/dc = 0.201)
was also synthesized by consecutive RAFT polymerizations using
CPADB as a CTA. To incorporate cell targeting, we selected the
neuron targeting peptide Tet1, which we previously have shown
to facilitate targeted transfection both in vitro and in vivo when
grafted to PEI.15 An N-terminus maleimide-functionalized Tet1
was conjugated by Michael-type addition to the terminal free
thiols of P(OEGMA)15-SS-P(GMA-TEPA)50 and P-
(OEGMA)15-b-P(GMA-TEPA)50 that were generated by
aminolysis of the dithioester end group during TEPA
functionalization. Tet1 functionalization efficiency was ∼33%,
likely due to competing thiolactone formation16 (see SI).
DNA binding of polymers was investigated by agarose gel
retardation assay. The results (Figure S5) indicate that all the
polymers exhibit similar DNA condensation above an N/P
(amino to phosphate) ratio of 4−5, and Tet1 peptide
conjugation does not significantly affect DNA binding of block
copolymers. The morphologies of the five polyplex formulations
were visualized by TEM (Figure 2a) at an N/P of 5. All materials
condensed plasmid DNA into compact particles with diameter
<50 nm. Polyplexes formed using block copolymers were more
compact than those formed by the P(GMA-TEPA)50 homopol-
ymer, and polyplexes containing Tet1 modification were more
polydisperse. The uniformity of polyplex morphology and size
might be improved in future work by controlling formulation, e.g.
by slow mixing or microfluidics-facilitated mixing17 as opposed
to the bulk mixing used here.
Figure 2. (a) TEM images of polyplexes formed by (a1) P(OEGMA)15-
b-P(GMA-TEPA)50, (a2) Tet1-P(OEGMA)15-b-P(GMA-TEPA)50,
(a3) P(OEGMA)15-SS-P(GMA-TEPA)50, (a4) Tet1-P(OEGMA)15-
SS-P(GMA-TEPA)50, and (a5) P(GMA-TEPA)50 at an N/P ratio of 5
(scale bar: 200 nm). (b) Change in the size of various polyplexes as
measured by DLS at 37 °C in the presence of 150 mM PBS, 150 mM
PBS + 10 mM DTT, and OptiMEM: (b1) P(OEGMA)15-b-P(GMA-
TEPA)50, (b2) Tet1-P(OEGMA)15-b-P(GMA-TEPA)50, (b3) P-
(OEGMA)15-SS-P(GMA-TEPA)50, (b4) Tet1-P(OEGMA)15-SS-P-
(GMA-TEPA)50. All the polyplexes were prepared at an N/P of 5.
minority populations of larger particles that skew the average
diameters measured by light scattering and also to measurement
in salt medium versus water. Only a slight increase in particle size
was observed for the polyplexes formed with the block
copolymers over 20 h following the addition of physiological
levels salt or Opti-MEM media, demonstrating excellent colloidal
stability of formed polyplexes. In contrast, polyplexes of P(GMA-
TEPA)50 homopolymer formed large aggregates with diameter
>1000 nm within 1 h under the same conditions (Figure S6c).
The overall results confirm that the 4.5 kDa P(OEGMA) block
provides sufficient extracellular colloidal stability for P(GMA-
TEPA)50 polyplexes. To investigate whether the polyplexes
formed using reducible block copolymers would be deshielded in
the cytosol to facilitate DNA release in the intracellular reducing
environment, the particle sizes of polyplexes in the presence of 10
mM dithiothreitol (DTT) were monitored over time. Polyplexes
of Tet1-P(OEGMA)15-SS-P(GMA-TEPA)50 and P-
(OEGMA)15-SS-P(GMA-TEPA)50 were found to increase in
size due to reduction-triggered deshielding, whereas polyplexes
formed from the nonreducible polymers were stable in size.
Hence, polyplexes of P(OEGMA)15-SS-P(GMA-TEPA)50 may
have excellent colloidal stability in the circulation and still be
readily destabilized in the cell cytosol, facilitating DNA release.
The in vitro transfection efficiency of reducible and non-
reducible polyplexes was evaluated in four different cell lines
HeLa, HEK293T, HepG2, and 2-day differentiated PC-12 cells
by luciferase assayusing P(GMA-TEPA)50 as a control. Figure
3a summarizes the transfection data for these cell lines. The
reversibly shielded polyplexes formed by P(OEGMA)15-SS-
P(GMA-TEPA)50 mediated significantly higher transfection
efficiency in all four cell lines compared to the stably shielded
analogue under identical conditions, affording up to 10 times
higher transfection efficiency depending on the cell type. The
results confirm that the reducible disulfide bond can improve
Poor salt stability has been a significant obstacle for in vivo
application of unshielded polyplexes.18,19 The salt stability of
polymer/DNA complexes was studied in both PBS (150 mM,
pH 7.4) and Opti-MEM using dynamic light scattering (DLS,
Figure 2b; see Figure S6 for full kinetics). The larger size
measured by DLS compared to TEM might be attributed to
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ASSOCIATED CONTENT
* Supporting Information
Experimental details and characterization data. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
spun@u.washington.edu
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Work funded by NIH 1R01NS064404. D.S.H.C. was supported
by NIH training grant T32CA138312. We thank Julie Shi and
Selvi Srinivasan for technical help and scientific discussion.
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Figure 3. (a) Transfection efficiency of polyplexes based on
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