Substrate-initiated synthesis of cell-penetrating poly(disulfide)s

Article abstract of DOI:10.1021/ja311961k

Lessons from surface-initiated polymerization are applied to grow cell-penetrating poly(disulfide)s directly on substrates of free choice. Reductive depolymerization after cellular uptake should then release the native substrates and minimize toxicity. In the presence of thiolated substrates, propagators containing a strained disulfide from asparagusic or, preferably, lipoic acid and a guanidinium cation polymerize into poly(disulfide)s in less than 5 min at room temperature at pH 7. Substrate-initiated polymerization of cationic poly(disulfide)s and their depolymerization with dithiothreitol causes the appearance and disappearance of transport activity in fluorogenic vesicles. The same process is further characterized by gel-permeation chromatography and fluorescence resonance energy transfer.

Full text of DOI:10.1021/ja311961k

Communication
pubs.acs.org/JACS
Substrate-Initiated Synthesis of Cell-Penetrating Poly(disulfide)s
Eun-Kyoung Bang,† Giulio Gasparini,† Guillaume Molinard, Aurelien Roux, Naomi Sakai,
́
and Stefan Matile*
School of Chemistry and Biochemistry, University of Geneva, Geneva, Switzerland
S
* Supporting Information
ABSTRACT: Lessons from surface-initiated polymeriza-
tion are applied to grow cell-penetrating poly(disulfide)s
directly on substrates of free choice. Reductive depolyme-
rization after cellular uptake should then release the native
substrates and minimize toxicity. In the presence of
thiolated substrates, propagators containing a strained
disulfide from asparagusic or, preferably, lipoic acid and a
guanidinium cation polymerize into poly(disulfide)s in less
than 5 min at room temperature at pH 7. Substrate-
initiated polymerization of cationic poly(disulfide)s and
their depolymerization with dithiothreitol causes the
appearance and disappearance of transport activity in
fluorogenic vesicles. The same process is further
characterized by gel-permeation chromatography and
fluorescence resonance energy transfer.
ell-penetrating peptides (CPPs) are short, polycationic
peptides or protein domains that are used by viruses to
C
enter cells.1,2 Their unique ability to transport linked substrates
across lipid bilayer membranes has attracted great interest in
biomedical applications. Substrates of varying sizes and
properties, e.g., small fluorophores to proteins and quantum
dots, have been successfully transported into cells using CPPs.
The mechanism of cellular uptake is under debate, currently
favored are endocytosxis (i.e., macropinocytosis) or passive
diffusion across the membrane, depending on conditions.
Multiple, moderately hydrophobic cations seem to be all that is
needed. Guanidinium cations, as in arginine, are most common,
alternatives include ammonium or phosphonium cations.1 The
originally peptidic oligomer backbone has been extensively
varied, covering oligocarbamates, β-peptides and several
variations of synthetic polymers.1 Currently, cell-penetrating
poly(disulfide)s are emerging as the cell-penetrating molecules
of the future because their cytosolic degradation liberates the
substrate and eliminates toxicity, one of the key disadvantages
associated with CPPs.3−5 However, cell-penetrating poly-
(disulfide)s have so far been used mainly in noncovalent
polyplexes for gene transfection, and covalent attachment of
substrates would be difficult with their preparation methods.
We have found recently that poly(disulfide)s can be grown
directly on solid substrates by surface-initiated ring-opening
disulfide-exchange polymerization.6 Therefore, we wondered
whether the same methodology could be used to prepare cell-
penetrating poly(disulfide)s with covalently attached substrates
in solution (Figure 1). Probes or drugs that contain thiol group
but cannot penetrate cells without assistance are the ideal
substrates, which could serve as an initiator to be appended
Figure 1. For the covalent delivery of unmodified probes,
guanidinium-containing propagators (e.g., 1−4) are polymerized on
thiolated substrates (e.g., 5−7) and terminated with iodoacetamides
(e.g., 8, 9). After uptake, cell-penetrating poly(disulfide)s are degraded
by reductive depolymerization to eliminate toxicity and release the
unmodified substrate.
with a membrane-active poly(disulfide). Thiolated siRNA, for
instance, is commercially available. The generality of this
approach promises a conceptually innovative solution for a
central current challenge, i.e., the noninvasive, nontoxic delivery
of unmodified substrates in well-defined, covalent systems
rather than complex, noncovalent formulations. In this initial
report on the topic, we describe the design, synthesis and
evaluation of propagators for the substrate-initiated synthesis of
cell-penetrating poly(disulfide)s. Their formation in less than 5
min at pH 7 and their depolymerization with 10 mM
dithiothreitol (DTT) can be followed directly as appearance
and disappearance of transport activity in fluorogenic vesicles.
To ultimately combine surface-initiated polymerization6 with
cellular uptake,1−5 we prepared the strained disulfides 1−4 as
possible propagators, thiols 5−7 as initiators, and the
iodoacetamides 8 and 9 as terminators (Figure 1). The
Received: December 7, 2012
Published: January 30, 2013
© 2013 American Chemical Society
2088
dx.doi.org/10.1021/ja311961k | J. Am. Chem. Soc. 2013, 135, 2088−2091

Journal of the American Chemical Society
Communication
synthesis of all new compounds was straightforward and is
described in detail in the Supporting Information (SI Schemes
S1−S7, Figures S10−S23).7 Only freshly prepared material was
used.
Fluorogenic vesicles are convenient analytical tools to follow
reactions with minimal effort and maximal speed.8 Here, EYPC-
LUVs⊃CF, i.e., large unilamellar vesicles (LUVs) composed of
egg yolk phosphatidylcholine (EYPC) and loaded with 5(6)-
carboxyfluorescein (CF), were used. EYPC-LUVs⊃CF report
CF release as fluorescence recovery because local dilution
reduces self-quenching.
increase in activity upon substrate-initiated polymerization of
propagator 1 with initiator 5 and terminator 9 (Figures 2B, ●,
S2−S4).
According to activity in EYPC-LUVs⊃CF, substrate-initiated
polymerization of propagator 1 was accomplished in less than 5
min (Figure 3A, ●). Polymerization was better in the presence
than in the absence of initiator 5 (Figures 3A, S2−S4). This
conclusion was supported by gel-permeation chromatography
(GPC). Polymers obtained from propagator 1 in the presence
of initiator 5 were of high molecular weight (Mw = 62.7 kDa)
and dispersity (PDI = 1.83, Figure 3B, solid). Considering
increasing transport activity with polymer length but less
predictable length-dependence of cellular uptake of CPPs, high
molecular weight and dispersity compared to commercially
available polyarginine (Mw = 16.7 kDa, PDI = 1.7, Figure 3C,
solid) were both very desirable characteristics. The same was
true for the disappearance of all activity in EYPC-LUVs⊃CF
within minutes of incubation with 10 mM DTT (Figure S7).7
This is in the range of cytosolic glutathione (∼5 mM) and thus
confirms the previously reported biodegradability of cell-
penetrating poly(disulfide)s.3,4 Without initiator, weaker
absorbance, i.e., lower yield of polymer, at lower Mw ∼ 16.2
kDa was observed (PDI = 2.1, Figure 3B, dashed).
The addition of propagator 1 to EYPC-LUVs⊃CF caused CF
release only above a relatively high EC50 = 129.9
0.7 μM
(Figures 2A, B, ○, S1−S3, S6). Ring-opening disulfide
The polymerization of propagator 1 was analyzed systemati-
cally from pH 5 to pH 9 and concentrations from 25 to 200
mM in the presence and the absence of 5 mM initiator 5 (pKa
∼ 9.5, Figure 4A, B). Best results were obtained with 100 mM 1
in 1 M TEOA, pH 7 (Figure 4A, B, dotted lines). At lower pH
and concentrations, the substrate-initiate polymerization was
○
●
Figure 2. Activity of monomers ( ) and polymers ( ). (A) Change
in CF emission intensity I during the addition of reaction mixtures
with and without initiator 5 (50 s, 75 μM final guanidinium (monomer
1) concentration) and excess Triton X100 (300 s) to EYPC-
○
●
LUVs⊃CF. Reaction mixtures: 100 mM 1, 0 ( ) or 5 mM 5 ( ),
1 M TEOA, pH 7, 10 min; termination: 5 mM 9. (B) Transport
activity Y of 1 before ( ) and after polymerization ( , 5 mM 5) with
increasing concentration of guanidinium cations (Y = I before lysis in
A (∼5 min)).
○
●
exchange polymerization3 of 1 (100 mM, pH 7, 1 M
triethanolamine (TEOA) buffer), initiated with thiol 5 (5
mM), was followed by adding aliquots of the reaction mixture
to EYPC-LUVs⊃CF after termination with iodoacetamide 9.
Rapid fluorescence recovery was observed with increasing
reaction time (Figure 3A, ●). At saturation, dose response
curves were recorded for the obtained polymers (Figures 2A, B,
●, S1−S4). An EC50 = 3.2 1.6 μM calculated to a 40-fold
Figure 4. Dependence on propagator concentration (A), pH (B) and
initiator concentration (C, D). (A) YP after polymerization of
●
○
increasing concentration c of 1 with ( ) and without 5 ( , 5 mM,
1 M TEOA, pH 7, 10 min; assay: 75 μM guanidinium each, YP = Y
normalized to Y = 0 before polymerization and Y = 1 for maximal
●
○
Figure 3. Polymers of 1 made with ( , solid) and without ( ,
dashed) initiator 5. (A) Y during polymerization of 1 (100 mM) with
●
○
●
( ) and without 5 ( , 5 mM, pH 7). (B) GPC of 1 (100 mM)
polymerized with (solid) and without 5 (dashed, 5 mM, pH 7),
compared to (C) polyarginine (top) and standards (bottom, MW 43,
25, 13.7, and 6.5 kDa); Superdex 75, 30% acetonitrile in acetate buffer,
pH 6.5.
activity). (B) Y after polymerization with increasing pH (5 mM 5 ( ),
0 mM 5 ( ), 100 mM 1, 1 M buffer). (C) Y after polymerization with
7
○
increasing concentration of 5 (100 mM 1, pH 7). (D) GPC after
polymerization with 5 (with increasing tR: 0.5, 1, 2, 5, 10 mM; 200
mM 2, pH 7.5).
2089
dx.doi.org/10.1021/ja311961k | J. Am. Chem. Soc. 2013, 135, 2088−2091

Journal of the American Chemical Society
Communication
incomplete, at higher pH, random polymerization without
initiator as well as precipitation started to interfere.
In summary, substrate-initiated polymerization of cell-
penetrating poly(disulfide)s is introduced as a conceptually
new approach to cellular uptake. Two types of propagators and
four unrelated methods to prove direct growth of polymers on
substrates are described. Namely, (a) polymers obtained with
and without initiators are different, (b) the dependence on
initiator concentration is bell-shaped, (c) labeled initiators are
eluted with polymers in GPC, and (d) FRET between donating
initiators and accepting terminators decreases with polymer-
ization time. Ring-opening disulfide exchange polymerization
with propagators derived from lipoic acid is facile to control
(pH, concentration of initiators, propagators, etc.) and gives
polymers with high, stimuli-responsive transport activity in
neutral lipid bilayers, whereas propagators derived from
asparagusic acid are too reactive and give polymers that require
counterion activation for function. With these complete, clear
and consistent results, the newly introduced system is ready for
cellular uptake experiments10 and copolymerization studie-
s1a,6b,f to modulate the properties of the cell-penetrating
poly(disulfide)s.
The bell-shaped dependence on initiator concentration was
in agreement with the formation of fewer polymers at low and
more but shorter and thus less active ones at high initiator
concentrations (Figure 4C). Corroborative evidence for the
incorporation of the initiators into the polymers was obtained
by GPC. Polymers obtained from propagator 2 and increasing
concentrations of initiator 5 gave polymers with decreasing
molecular weight and dispersity (Figure 4D). Moreover,
polymers obtained with Cys-Trp initiator 6 showed the
tryptophan emission in the GPC peak. The relative Trp
emission increased with decreasing molecular weight, that is
decreasing polymer/initiator ratio (Figure S8).7
The substrate-initiated polymerization of propagators 1 and
2 with the strained disulfides from lipoic acid was
straightforward to control and optimize. The disulfides from
asparagusic acid are ideal for surface-initiated polymerization6
but turned out to be too reactive9 for substrate-initiated
polymerization in solution. Independent of their backbone,
propagators 3 and 4 more easily polymerized with less
difference between substrate-initiated and random polymer-
ization without initiator (Figure S6). Moreover, cell-penetrating
poly(disulfide)s obtained from lipoyl propagators 1 and 2 were
active in EYPC LUVs, whereas the less lipophilic polymers
from asparagusyl propagators 3 and 4 were poorly active.
However, like arginine-rich CPPs,2 all polymers could be
activated in EYPC LUVs by counterions such as pyrenebutyrate
(Figures S5, S6).7
ASSOCIATED CONTENT
* Supporting Information
Details on experimental procedures. This material is available
free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
Stefan.Matile@unige.ch
■
To probe for substrate-initiated polymerization also with the
less perfect asparagusyl propagators, fluorescence resonance
energy transfer (FRET) was considered as a method
complementary to the functional studies with fluorogenic
LUVs and GPC described above for the preferable lipoyl
propagators. Polymerization of propagator 4 in CHCl3/DMF
3:1 was initiated with the yellow, green-fluorescent naphthale-
nediimide (NDI)6 fluorophore 7 (λex = 469 nm, λem = 484 nm)
Author Contributions
†These authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank D. Jeannerat, A. Pinto and S. Grass for NMR
measurements, the Sciences Mass Spectrometry (SMS) plat-
form for mass spectrometry services, and the University of
Geneva, the European Research Council (ERC Advanced
Investigator), the National Centre of Competence in Research
(NCCR) in Chemical Biology and the Swiss NSF for financial
support.
and 0.25% Hunig base (DIEA) as base, and terminated with the
̈
red NDI 8 (λex = 552 nm, λem = 582 nm, Figure 5). With
REFERENCES
■
(1) (a) Tezgel, A. O.; Gonzalez-Perez, G.; Telfer, J. C.; Osborne, B.
A.; Minter, L. M.; Tew, G. N. Mol. Ther. 2013, 21, 201−209. (b) Som,
A.; Reuter, A.; Tew, G. N. Angew. Chem., Int. Ed. 2012, 51, 980−983.
(c) Appelbaum, J. S.; LaRochelle, J. R.; Smith, B. A.; Balkin, D. M.;
Holub, J. M.; Schepartz, A. Chem. Biol. 2012, 19, 819−830.
́
(d) Ornelas-Megiatto, C.; Wich, P. R.; Frechet, J. M. J. Am. Chem.
Soc. 2012, 134, 1902−1905. (e) Treat, N. J.; Smith, D.; Teng, C.;
Flores, J. D.; Abel, B. A.; York, A. W.; Huang, F.; McCormick, C. L.
ACS Macro Lett. 2012, 1, 100−104. (f) Budhathoki-Uprety, J.; Peng,
L.; Melander, C.; Novak, B. M. ACS Macro Lett. 2012, 1, 370−374.
(g) Song, A.; Walker, S. G.; Parker, K. A.; Sampson, N. S. ACS Chem.
Biol. 2011, 6, 590−599. (h) Tezgel, A. O.; Telfer, J. C.; Tew, G. N.
Biomacromolecules 2011, 12, 3078−3083. (i) Gorska, K.; Manicardi, A.;
Barluenga, S.; Winssinger, N. Chem. Commun. 2011, 47, 4364−4366.
(k) Dix, A. V.; Fischer, L.; Sarrazin, S.; Redgate, C. P. H.; Esko, J. D.;
Tor, Y. ChemBioChem 2010, 11, 2302−2310. (l) Inomata, K.; Ohno,
A.; Tochio, H.; Isogai, S.; Tenno, T.; Nakase, I.; Takeuchi, T.; Futaki,
S.; Ito, Y.; Hiroaki, H.; Shirakawa, M. Nature 2009, 458, 106−109.
(m) McNaughton, B. R.; Cronican, J. J.; Thompson, D. B.; Liu, D. R.
Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 6111−6116. (n) Trantow, B.
Figure 5. FRET from initiator 7 to terminator 8. Emission spectra (λex
= 445 nm, CHCl3) after polymerization of 4 (25 mM) with 7 (1 mM)
for 5 s (black), 30 s (blue) and 60 s (red) in CHCl3/DMF 3:1 (0.25%
DIEA), terminated with 8 (2 mM, solid) and depolymerized with
DTT (10 mM, dashed).
increasing polymerization time, the FRET emission at λem
=
582 nm in CHCl3 decreased (Figure 5). This decrease was
consistent with increasing distance between initiator and
terminator with increasing reaction time. Depolymerization
with DTT caused nearly complete disappearance of FRET
(Figure 5, dotted). These results further demonstrated the
incorporation of the terminator in the polymer.
2090
dx.doi.org/10.1021/ja311961k | J. Am. Chem. Soc. 2013, 135, 2088−2091

Journal of the American Chemical Society
Communication
M.; Cooley, C. B.; Nederberg, F.; Kiesewetter, M. K.; Hedrick, J. L.;
Waymouth, R. M.; Wender, P. A. J. Am. Chem. Soc. 2009, 131, 16401−
16403. (o) Kolonko, E. M.; Pontrello, J. K.; Mangold, S. L.; Kiessling,
L. L. J. Am. Chem. Soc. 2009, 131, 7327−7333. (p) Wender, P. A.;
Galliher, W. C.; Goun, E. A.; Jones, L. R.; Pillow, T. H. Adv. Drug
Delivery Rev. 2008, 60, 452−472. (q) Esbjorner, E. K.; Lincoln, P.;
Norden, B. Biochem. Biophys. Acta 2007, 1768, 1550−1558.
(r) Shimanouchi, T.; Walde, P.; Gardiner, J.; Mahajan, Y. R.;
Seebach, D.; Thomae, A.; Kraemer, S. D.; Voser, M.; R. Kuboi, R.
Biochem. Biophys. Acta 2007, 1768, 2726−2736. (s) Kale, A. A.;
Torchilin, V. P. Bioconjugate Chem. 2007, 18, 363−370. Martinell, M.;
́ ́
Salvatella, X.; Fernandez-Carneado, J.; Gordo, S.; Feliz, M.; Menendez,
M.; Giralt, E. ChemBioChem 2006, 7, 1105−1113. (t) Schmidt, D.;
Jiang, Q. X.; MacKinnon, R. Nature 2006, 444, 775−779. (u) Fischer,
R.; Fotin-Mleczek, M.; Hufnagel, H.; Brock, R. ChemBioChem 2005, 6,
2126−2142. (v) Pantarotto, D.; Briand, J.-P.; Prato, M.; Bianco, A.
Chem. Commun. 2004, 16−17. (w) Jiang, T.; Olson, E. S.; Nguyen, Q.
T.; Roy, M.; Jennings, P. A.; Tsien, R. Y. Proc. Natl. Acad. Sci. U.S.A.
2004, 101, 17867−17872. (x) Umezawa, N.; Gelman, M. A.; Haigis,
M. C.; Raines, R. T.; Gellman, S. H. J. Am. Chem. Soc. 2002, 124, 368−
369.
(2) (a) Sakai, N.; Matile, S. J. Am. Chem. Soc. 2003, 125, 14348−
14356. (b) Takeuchi, T.; Kosuge, M.; Tadokoro, A.; Sugiura, Y.; Nishi,
M.; Kawata, M.; Sakai, N.; Matile, S.; Futaki, S. ACS Chem. Biol. 2006,
1, 299−303.
(3) Bang, E.-K.; Lista, M.; Sforazzini, G.; Sakai, N.; Matile, S. Chem.
Sci. 2012, 3, 1752−1763.
(4) (a) Kim, T. I.; Rothmund, T.; Kissel, T.; Kim, S. W. J. Controlled
Release 2011, 152, 110−119. (b) Kim, T.; Kim, S. W. React. Funct.
Polym. 2011, 71, 344−349. (c) Lin, C.; Engbersen, J. F. J. Expert Opin.
Drug Delivery 2009, 6, 421−439. (d) Son, S.; Namgung, R.; Kim, J.;
Singha, K.; Kim, W. J. Acc. Chem. Res. 2011, 152, 110−119. (e) Meng,
F.; Hennink, W. E.; Zhong, Z. Biomaterials 2009, 30, 2180−2198.
(5) Thiols at the cell surface have been proposed to further
contribute to the uptake of disulfide-containing systems. Torres, A. G.;
Gait, M. J. Trends Biotechnol. 2012, 30, 185−190.
(6) (a) Sakai, N.; Lista, M.; Kel, O.; Sakurai, S.; Emery, D.; Mareda,
J.; Vauthey, E.; Matile, S. J. Am. Chem. Soc. 2011, 133, 15224−15227.
(b) Lista, M.; Areephong, J.; Sakai, N.; Matile, S. J. Am. Chem. Soc.
2011, 133, 15228−15230. (c) Sakai, N.; Matile, S. J. Am. Chem. Soc.
2011, 133, 18542−18545. (d) Charbonnaz, P.; Sakai, N.; Matile, S.
Chem. Sci. 2012, 3, 1492−1496. (e) Areephong, J.; Orentas, E.; Sakai,
N.; Matile, S. Chem. Commun. 2012, 48, 10618−10620. (f) Orentas,
E.; Lista, M.; Lin, N.-T.; Sakai, N.; Matile, S. Nat. Chem. 2012, 4, 746−
750.
(7) See SI.
(8) (a) Takeuchi, T.; Matile, S. Chem. Commun. 2013, 49, 19−29.
(b) Litvinchuk, S.; Tanaka, H.; Miyatake, T.; Pasini, D.; Tanaka, T.;
Bollot, G.; Mareda, J.; Matile, S. Nat. Mater. 2007, 6, 576−580.
(c) Sakai, N.; Sorde,
́
N.; Matile, S. J. Am. Chem. Soc. 2003, 125, 7776−
7777. (d) Sorde, N.; Das, G.; Matile, S. Proc. Natl. Acad. Sci. U.S.A.
́
2003, 100, 11964−11969. (e) Das, G.; Talukdar, P.; Matile, S. Science
2002, 298, 1600−1602.
(9) Burns, J. A.; Whitesides, G. M. J. Am. Chem. Soc. 1990, 112,
6296−6303.
(10) Preliminary results with cell-penetrating poly(disulfide)s grown
with 1 and 2 on fluorescent probes as initiators revealed efficient
uptake into HeLa cells. These ongoing studies will be reported in due
course.
2091
dx.doi.org/10.1021/ja311961k | J. Am. Chem. Soc. 2013, 135, 2088−2091