Multifunctional nanoworms and nanorods through a one-step aqueous dispersion polymerization

Article abstract of DOI:10.1021/ja500092m

Producing synthetic soft worm and rod structures with multiple chemical functionalities on the surface would provide potential utility in drug delivery, nanoreactors, tissue engineering, diagnostics, rheology modifiers, enzyme mimics, and many other applications. Here, we have synthesized multifunctional worms and rods directly in water using a one-step reversible addition-fragmentation chain transfer (RAFT)-mediated dispersion polymerization at high weight fractions of polymer (>10 wt %). The chain-end functionalities included alkyne, pyridyl disulfide, dopamine, β-thiolactone, and biotin groups. These groups could further be converted or coupled with biomolecules or polymers. We further demonstrated a nanorod colorimetric system with good control over the attachment of fluorescent probes.

Full text of DOI:10.1021/ja500092m

Communication
pubs.acs.org/JACS
Multifunctional Nanoworms and Nanorods through a One-Step
Aqueous Dispersion Polymerization
Zhongfan Jia, Valentin A. Bobrin, Nghia P. Truong, Marianne Gillard, and Michael J. Monteiro*
Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, Brisbane QLD 4072, Australia
S
* Supporting Information
polymers with controlled diameters and lengths.12 Although the
ABSTRACT: Producing synthetic soft worm and rod
structures with multiple chemical functionalities on the
surface would provide potential utility in drug delivery,
nanoreactors, tissue engineering, diagnostics, rheology
modifiers, enzyme mimics, and many other applications.
Here, we have synthesized multifunctional worms and rods
directly in water using a one-step reversible addition−
fragmentation chain transfer (RAFT)-mediated dispersion
polymerization at high weight fractions of polymer (>10
wt %). The chain-end functionalities included alkyne,
pyridyl disulfide, dopamine, β-thiolactone, and biotin
groups. These groups could further be converted or
coupled with biomolecules or polymers. We further
demonstrated a nanorod colorimetric system with good
control over the attachment of fluorescent probes.
self-assembly technique has found potential in many biomedical
and other applications, the technique requires considerable time
to produce the nanostructure of choice, the chemical
functionality at the surface can become buried within the shell
during the cross-linking procedure, the weight fraction of
polymer is ∼0.5 wt %, and each chain-end functional polymer
must be separately synthesized13 which can influence the self-
assembly process. Currently, there is no direct polymerization
procedure to create stable worms and rods directly in water at
high weight fractions of polymer with multifunctional and
orthogonal chemical groups on the surface through a one-step
process. Producing such nanostructures directly in water at
weight fractions of 10−20 wt % would provide a facile method to
covalent attach bio- and other molecules that mimic features of
natural systems (e.g., worm- and rod-like viruses) and, therefore,
significantly broaden the potential of such nanostructures as
drug,14 siRNA15 and vaccine16 delivery vehicles, organic
photonics,17 diagnostics, nanoreactors to self-healing coatings.18
Herein, we report a method to produce multifunctional worms
directly in water using a one-step reversible addition−
fragmentation chain transfer (RAFT)-mediated dispersion
polymerization (Scheme 1). End-group functional poly(N-
isopropylacrylamide) (PNIPAM) thermoresponsive Macro-
CTAs in the presence of styrene (STY), AIBN, and SDS were
first assembled in water at 70 °C (i.e., well above their lower
critical solution temperature (LCST) of PNIPAM) and
polymerized for 3.5 h to form diblock copolymers of PNIPAM
and PSTY at a weight fraction of 10 wt %. The latex solution was
cooled to 23 °C (i.e., below the LCST), at which temperature the
PNIPAM blocks became water-soluble, transforming the
spherical structures into multifunctional nanoworms with a
diameter of ∼15 nm and lengths ranging between 2 and 5 μm in
the presence of toluene (20 μL per 1 mL of latex solution) as
plasticizer. The advantage of this process is that the multifunc-
tional chemical groups are located at the surface of the structures,
maximizing further chemical transformations and coupling
reactions. This technique, denoted as the temperature-directed
morphology transformation (TDMT), has allowed a wide range
of nanostructures to be produced,19 ranging from spheres,
worms, vesicles, donuts, and lamella sheets; structures that were
not only stable in water for long periods of time (e.g., the worms
were stable for a year at room temperature) but also could be
freeze-dried and rehydrated without structural reorganization.
ultifunctional nanostructures, including spheres, vesicles,
Mworms and rods, surface functionalized with combina-
tions of biological molecules (e.g., siRNA, peptides, enzymes),
prodrugs, imaging agents, and other molecules have generated
intense interest for a wide variety of applications.1 Long and
flexible worms have in vivo blood circulation times about 10 times
longer than their spherical analogues2 and have been found to
have significantly longer circulation times than any synthetic
particle or PEO-coated stealth vesicles.3 Short rods, on the other
hand, have much shorter circulations times but are more
efficiently taken up by cells.2 This together with the excellent in
vivo antitumor activity3 demonstrate the great potential of worms
and rods in nanomedicine. The unique properties of worms and
rods is not surprising as nature has produced the well-known
cylindrical biostructured viruses to elude the body’s immune
system, which include the worm-like Potato Virus Y (PVY)4 and
rod-like Tobacco Mosaic Virus (TMV).5 Worms have further
attracted interest due to their high aspect ratio to alter the
rheology6 and gelation properties of materials.7
The most utilized method to prepare worms is through the
self-assembly of amphiphilic diblock copolymer, starting from a
good solvent for both blocks followed by the slow addition of
water (a poor solvent for hydrophobic block) until reaching the
required solvent ratio.8 The worms are cross-linked either in the
core or shell9 depending upon the application10 and extensively
dialyzed with water to remove any organic solvent and unreacted
cross-linker. This method has allowed chemical functionality on
the surface of spheres and other structures for coupling with
sugars, folic acid, oligonucleotides, and other biomolecules.11
Worms and rods can also be prepared from dendronized
Received: January 8, 2014
© XXXX American Chemical Society
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Scheme 1. Synthesis of Multifunctional Nanoworms and Nanorods Through RAFT-Mediated Emulsion Polymerization in Water
Followed by TDMT and Ultrasound Cutting
Six chain-end functional RAFT agents were synthesized
through modification of the carboxylic acid group on the
trithiolcarbonate RAFT agent, 2-(((butylthio)carbonothioyl)-
thio)-2-methylpropanoic acid (see Experimental in SI). The
functional groups chosen here consisted of an alkyne (Alk) for
the copper catalyzed alkyne−azide cycloaddition (CuAAC)
‘click’ reaction,20 pyridyl disulfide (PDS) for thiol-disulfide
exchange or thiol−ene reactions,21 dopamine (Dopa) for specific
binding to metal surface,22 β-thiolactone (Tla) for amination
reaction23 and biotin for binding to streptavidin.24 The RAFT-
mediated polymerization of these RAFT agents with NIPAM
produced six functional MacroCTAs with number-average
molecular weights (Mn’s) ranging from 5100 to 5510 (i.e., with
the functional groups were located at the PNIPAM chain-ends
(see Scheme 1 for transmission electron micrograph (TEM) of
the worms). We then utilized the Winnik et al.25 ultrasound
method to cut the worms to short rods with an approximate
length of 150 nm and the same core diameter (see TEM in
Scheme 1 and histogram in Figure S18). Our ultrasound
conditions for the cutting of worms supported the theory of
Winnik et al.25 that the limiting rod size was 100 nm (see Figure
S19). Our procedure demonstrates the ease of producing worms
and rods with many different functional groups on the surface via
a one-step polymerization directly in water and at high weight
fractions of polymer. NMR analysis of this and the following
block copolymers were freeze-dried to remove all water and low
boiling point compounds and then dissolved in CDCl3, and for
SEC analysis a small amount of latex was dissolved in THF and
directly injected into the SEC system.
1
∼44 repeating units) as determined by H NMR and with
polydispersity indexes (PDISEC’s) all below 1.12 (see Table S1,
1
SEC chromatograms and H NMR spectra in SI), suggesting
excellent control over the molecular weight distribution (MWD)
and maintenance of high chain-end fidelity.
The next polymerization (Table S2, expt 1) used 100% of the
alkyne MacroCTA (R2) to produce a narrow particle size
distribution (Dh = 189 nm and PDIDLS = 0.067) and narrow
MWD (Mn = 8500, PDISEC = 1.14). Using the same TDMT
procedure above, the spherical latex particles were transformed
to worms and then cut to short rods using ultrasound (see Figure
S17), in which each PNIPAM chain-end consisted of a reactive
alkyne moiety (Figure 1A). The worms and rods were coupled
through the CuAAC ‘click’ reaction with PNIPAM-N3 and PEG-
N3, (see SI for experimental and characterization details for these
azide functional polymers). The coupling efficiency determined
by 1H NMR for PNIPAM-N3 (Mn = 4340, PDISEC = 1.06) and
PEG-N3 (Mn = 2800, PDISEC= 1.10) to the long worms was 88
and 82%, respectively, and coupling efficiency to the short rods
was slightly higher at 86 and 90%, respectively. Heating these
worm and rod solutions above the LCST (∼37 °C) of the
PNIPAM block produced a gel that when cooled dissociated
back to a sol; a process that is fully reversible (Figure 1B
depicting the gel−sol transformation). The alkyne functional
The first RAFT-mediated dispersion polymerization consisted
of mixing four different MacroCTAs (i.e., R1, R2, R3, and R4 in
Scheme 1 at a ratio of 0.4:0.2:0.2:0.2) with styrene, SDS, and
AIBN at 70 °C (Table S2, expt 7). After a polymerization time of
∼3.5 h (∼75% conversion), the resultant emulsion produce
polymer particles measured at 70 °C with an average number-
average diameter (Dh) of 210 nm and a variance (i.e., PDIDLS) of
0.115, suggesting a relatively narrow particle size distribution.
The MWD of the resultant polymers was well-controlled with Mn
close to the theoretically calculated value and with a PDISEC of
1.10. The excellent control over the MWD further suggested
high chain-end fidelity (see SEC chromatograms in SI). A small
amount of toluene (20 μL per 1 mL of latex solution) was added
and immediately cooled to 15 °C. The toluene was added to
plasticize the glassy PSTY and aid in the temperature-directed
morphology transformation. Long and flexible worms formed
consisting of a PSTY core and a PNIPAM hairy layer, in which
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Figure 2. (A) Schematic showing the modification of 50% PDS
functional nanoworms and nanorods (Table S2, expt 2) with L-
glutathione (GSH). (B) Conjugation efficiency vs time for worm (blue
triangle) and rods (red circle) from the UV−vis spectra of released 2-
mercaptopyridine after the conjugation of GSH with PDS functional
nanoworms and nanorods, respectively; the molar ratio of GSH to PDS
was 5. (C) Schematic showing the modification of 50% Tla functional
nanoworms and nanorods (Table S2, expt 4) with allylamine and DPDS.
(D) UV−vis spectra showing the release of 2-mercaptopyridine for the
reaction of nanoworms (dark-red line) and nanorods (light-blue line)
with allylamine and DPDS.
Figure 1. (A) Schematic showing the modification of 100% alkyne
functional worms and rods R1-P(NIPAM45-b-STY39) (Table S2, expt 1)
through CuAAC ‘click’ reaction. (B) Photos showing the typical gel−sol
transformation. (C) Minimum weight percentage of gel formation at 37
°C: (a) R1-P(NIPAM45-b-STY39), (b) P(NIPAM43-b-NIPAM45-b-
STY39), and (c) P(EG45-b-NIPAM45-b-STY39); blue bar for worms
and red bar for rods.
worms and rods formed gels at a minimum weight fraction of 8
and 16 wt %, respectively (Figure 1C). Coupling PNIPAM-N3
significantly reduced the minimum wt% for gelation to 1.5 and 2
wt % for the worms and rods, respectively. This suggests that
above the LCST, the higher molecular weight of the second
PNIPAM block resulted in greater interworm interaction thus
significantly lowering the gel point. On the other hand, coupling
PEG-N3 increased the gel point, most probably due to the
decreased interactions between the worms or rods. This level of
control over the gel properties together with the variety of
chemical groups has the potential as scaffolds for tissue
engineering.
Taken together, the results demonstrate the efficient coupling
due to the location of the functional end groups extending from
the surface of the worms and rods well into the water phase.
To test the level of control over the percentage of functional
groups on the surface, we used three MacroCTAs (R1, R3, and
R6) to produce rods with different ratios of functional groups
(Table S2, expts 8−10) as shown in Figure 3. The rods were then
Other functional worms and rods were produced using the
TDMT method with pyridyl disulfide (PDS) or β-thiolactone
(Tla) end groups (Table S2, expts 2 and 4). Pyridyl disulfide has
been widely used in conjugation reactions of polymers and
biomolecules with sugar,26 peptides,27 oligonucleotides,28
proteins,29 and anticancer drugs.30 The worms and rods with
PDS (Table S2, expt 2) were prepared from an equal ratio of
MacroCTAs, R1, and R3. The MWD was again well-controlled,
and the worms and rods then characterized by TEM (Figure
S17). The PDS-worms and PDS-rods were further reacted with
the model peptide that is reduced L-glutathione (GSH, 5 equiv to
PDS groups) to produce peptide surface coated worms and rods
(Figure 2A). The excess of GSH was easily dialyzed out. The
conjugation efficiency to the nanorods was also measured by
UV−vis absorbance of 2-mercaptopyridine released after
coupling, giving a conjugation efficiency of 79%, which was
supported by the conjugation efficiency determined by 1H NMR
of 75% (through the loss of the pyridyl disulfide). The
conjugation efficiency was slightly lower for the worms (Figure
2B). In the case of the Tla worms and rods, we ring-opened the
thiolactone with allylamine and subsequently protected the
resultant free thiol using dipyridyl disulfide (DPDS) to form
pyridyl disulfide and alkene functional worms and rods in a one-
pot reaction (Figure 2C), allowing for the possibility of further
sequential reactions. The coupling efficiency with allylamine was
81% for worms and 95% for rods (see Table S4). The GHS and
allylamine are highly water soluble and should be effectively
dialyzed out before NMR or UV analysis. In addition, allylamine
has a low boiling point and will be removed after freeze drying.
Figure 3. Stoichiometric bioorthogonal conjugation of PDS and biotin
functional groups on the nanorods (Table S2, expts 8−10) with
maleimide functional Oregon green 488 and Streptavidin DyLight 550,
respectively. The ratio of SAv DyLight 550 to biotin was 1:4 and Oregon
green 488 maleimide to PDS was 1:1; (a−c) showing the colorimetric
change of nanorods after functionalization.
coupled through the PDS (R3) groups with the fluorescence
Oregon green 488 maleimide (1 equiv to PDS groups) and the
rods then further coupled with Streptavidin DyLight 550 (SAv
550) (0.25 equiv to biotin (R6) groups since there are four
binding sites on SAv 550). Confocal microscopy of the merged
image from the 488 and 550 nm channels showed that as the
biotin ratio to PDS was reduced from 2 to 0.5; the image changed
C
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Journal of the American Chemical Society
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ASSOCIATED CONTENT
■
S
* Supporting Information
Materials, synthetic procedures, characterization of compounds
and polymers. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
m.monteiro@uq.edu.au
Notes
The authors declare no competing financial interest.
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2425−2427.
ACKNOWLEDGMENTS
■
Australian Research Council (ARC) Discovery grant.
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