Simple but precise engineering of functional nanocapsules through nanoprecipitation

Article abstract of DOI:10.1002/anie.201402825

A general, rapid, and undemanding method to generate at will functional oil-filled nanocapsules through nanoprecipitation is reported. On the basis of polymer and hexadecane/water/acetone phase diagrams, the composition can be set so that polymer chains p

Full text of DOI:10.1002/anie.201402825

Angewandte
Chemie
DOI: 10.1002/anie.201402825
Functional Nanostructures
Simple but Precise Engineering of Functional Nanocapsules through
Nanoprecipitation**
Xibo Yan, Marco Delgado, Amy Fu, Pierre Alcouffe, Sꢀbastien G. Gouin, Etienne Fleury,
Joseph L. Katz, FranÅois Ganachaud, and Julien Bernard*
Abstract: A general, rapid, and undemanding method to
generate at will functional oil-filled nanocapsules through
nanoprecipitation is reported. On the basis of polymer and
hexadecane/water/acetone phase diagrams, the composition
can be set so that polymer chains preferentially stick at the
interface of the oil droplets to create nanocapsules. The
nanocapsules can be decorated with biorelevant molecules
(biotin, fluorescent tags, metal nanoparticles) within the shell
and loaded with hydrophobic molecules in a simple one-pot
procedure.
controlled radical polymerization under dispersion or emul-
sion polymerization conditions,[5] or 5) interfacial polymeri-
zation under miniemulsion conditions.[6]
A simple means of generating colloids relies on the so-
called nanoprecipitation technique,[7] the principle of which
lies in the supersaturation of whatever hydrophobic solute
(oil, polymer solids) primarily dissolved in a hydrophilic
solvent (e.g. acetone, THF) when a large excess of the
nonsolvent, water, is added. This technique, rediscovered just
over a decade ago and coined the “ouzo effect”, moved one
step forward with the establishment of well-defined phase
diagrams.[8] Whereas the generation of plain organic[9] or
polymeric[7b] nanoparticles is now well-documented (includ-
ing encapsulation in a polymeric matrix[10]), little has been
reported on the encapsulation of liquids by a polymer by
using the ouzo effect. This lack of studies on nanocapsule
generation surely lies in the difficulty to reproducibly
coprecipitate various substrates (oil, polymer, liposome…)
in a one-pot procedure
Herein, we present a general, undemanding method for
the construction of multifunctional, oil-filled nanocapsules
with controlled morphologies in a simple, straightforward
batch experiment. We demonstrate that a deep understanding
of phase diagrams allows delimitation of the window in which
narrowly dispersed core/shell nano-objects decorated with
biologically relevant species can be elaborated (see Figure 1).
More specifically, we demonstrate that in a narrow range of
polymer/water/acetone/hexadecane (HD) compositions, sol-
vent shifting forces the polymer chains to preferentially stick
at the interface of the oil droplets to generate polymeric
capsules in the full volume. We also show that the initial
incorporation in the organic phase of a diisocyanate[11] with
low water sensitivity both enables the structure of the
nanocapsules to be locked through cross-linking of the
polymer shell (Figure 1A) and the attachment of molecules
of interest primarily introduced in the water phase (Fig-
ure 1C,D). Nanocapsules can also be loaded with hydro-
phobic molecules (Figure 1D) or coated with metal nano-
particles (Figure 1B).
T
he generation of engineered synthetic capsules has
attracted considerable attention owing to their promising
application in the controlled release of pharmaceuticals,
catalysis, imaging, and biochemical reactions.[1] Whereas
microcapsule generation by chemical-engineering processes
has been possible for decades, routes to polymeric nano-
capsules require soft-matter technologies, such as: 1) well-
established self-organization of amphiphilic block copolymers
upon solvent displacement into polymersomes or micelles
possessing a cross-linkable shell and a core which can be
conveniently degraded,[2] 2) core removal of dendrimers,[3]
3) layer-by-layer deposition of polymers on sacrificial tem-
plate particles,[4] 4) polymerization-induced self-assembly by
[*] X. Yan, Dr. M. Delgado, P. Alcouffe, Prof. E. Fleury,
Dr. F. Ganachaud, Dr. J. Bernard
Universitꢀ de Lyon, 69003 Lyon (France)
and
INSA-Lyon, IMP, 69621 Villeurbanne (France)
and
CNRS, UMR 5223, Ingꢀnierie des Matꢀriaux Polymꢁres
69621 Villeurbanne (France)
E-mail: julien.bernard@insa-lyon.fr
Dr. A. Fu, Prof. J. L. Katz
Department of Chemical and Biomolecular Engineering
Johns Hopkins University
221 Maryland Hall, 3400 North Charles Street, Baltimore
MD 21218 (USA)
Dr. S. G. Gouin
LUNAM Universitꢀ, CEISAM Chimie et Interdisciplinaritꢀ, Synthꢁse,
Analyse, Modꢀlisation
UMR CNRS 6230, UFR des Sciences et des Techniques
2, rue de la Houssiniꢁre, BP 92208, 44322 Nantes Cedex 3 (France)
To exemplify this concept, we focus herein on the
production of functionalized, surfactant-free glyconanocap-
sules, which are promising candidates for drug-delivery
applications.[12] Although highly attractive, the development
of such glyco-nano-objects has been limited so far.[13] Water-
soluble glycopolymers of N-[7-(a-d-mannopyranosyloxy)-
heptyl]methacrylamide (HMM) were first prepared by poly-
merization mediated by 4-cyano-4-(phenylcarbonothioyl-
thio)pentanoic acid (see full details in the Supporting
Information). Polymerization reactions proceeded smoothly
[**] This research was supported by the Agence Nationale de la
Recherche (Programmes Blancs Starlet, ANR-12-BSV5-0016-01 and
LimOUzIne, ANR-10-BLAN-0942). X.Y. acknowledges the CSC for
a PhD grant. We thank Dr. Yann Bretonniꢁre and Dr. Stꢀphane
Chambert for discussions, and the CTm for microscopy analyses.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201402825.
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the resulting dispersions highlighted the presence of a single
population with a z-average diameter (Dz) of 124 nm and
a polydispersity index equal to 0.11 (see Figure S4).
To “freeze” the structure of the capsules and simulta-
neously promote their functionalization, a cross-linker, iso-
phorone diisocyanate (IPDI), was added initially to the
organic solution (Scheme 1). Figure 3 shows TEM pictures of
a series of nano-objects of increasing complexity. When HD
and PHMM were coemulsified without IPDI or with a low
content of diisocyanate (0.5 equiv per PHMM chain, 5.7 ꢀ
10À4 equiv per OH group), the structures did not survive to
TEM analysis (Figure 3A; see also Figure S5). HD leaked out
of the droplets, whereas most of the PHMM chains self-
assembled in small, dense dark dots of pure precipitated
glycopolymer (see also Figure S2). Upon the addition of
1 equivalent of IPDI per PHMM chain, the Dz value of the
capsules increased to 248 nm (PDI = 0.07), and the formation
of a thin, fragile, and deformable membrane was observed by
TEM at the surface of the droplets (Figure 3B). HD domains
and glycopolymer nanoparticles (from self-assembly of the
unreacted free chains during sample casting) were still
present. An increase in the IPDI content resulted in
progressive shrinkage of the capsule membranes as a result
of cross-linking reactions between PHMM chains, and the
Dz value of the capsules dropped to 200 and 150 nm upon the
addition of 5 and 24 equivalents, respectively, of IPDI (Fig-
ure 3C and Figure S5). The nano-objects also gradually
evolved to robust and perfectly spherical glyconanocapsules.
Accordingly, leakage of HD was suppressed, thus confirming
that the polymer membranes are sufficiently robust not to be
broken during the TEM process (casting/analysis). Outside
the defined f zone, nanocapsules cannot form (see Figure S6):
In domain b, for example, droplets are not covered by the
glycopolymer, so that, despite IPDI addition, free emulsion
Figure 1. General schematic illustration of functional nanocapsules
that can be built by nanoprecipitation: A) cross-linked oil-filled nano-
capsules; B) metal-nanoparticle-coated nanocapsules; C) nanocapsules
with bioactive molecules within the shell; D) core- or shell-tagged
fluorescent nanocapsules.
to yield two polymers (PHMM1 and PHMM2) exhibiting
molar masses of 24 and 102 kgmolÀ1 and a ꢁ value below 1.10
(see Table S1 and Figure S1 in the Supporting Information).
Phase diagrams for the polymers in water (solvent)/
acetone (nonsolvent) mixtures and for HD in acetone
(solvent)/water (nonsolvent) were then determined (see
Figures S2–S4 for the establishment of phase diagrams).
The cloud-point boundary of the polymer/water/acetone
ternary system was readily determined by the titration of
aqueous solutions of different polymer concentrations with
the step-by-step addition of acetone until the polymer
precipitated. It can basically be described by
a straight line at a constant mass fraction of
acetone (see Figure 2). The boundary is
slightly shifted to a lower mass fraction of
the solvent when titrations are performed with
the polymer possessing the highest molar mass
(see Figure S2). The phase diagram of HD,
superimposed on that of the polymerin
Figure 2 shows three apparent zones(for the
full diagram, see Figure S3). Above the bino-
dal curve, HD is perfectly miscible with the
acetone/water mixture (domains c and d). In
the ouzo domain (b), metastable emulsions
are formed, and for the largest concentrations
of HD, that is, beyond the “ouzo limit”
(domains a and e), the oil rapidly demixes.[14]
The requirement for nanocapsule forma-
tion is that emulsion droplets are generated
and adsorption of the polymer at the interface
occurs. The domain in which this process
Figure 2. Overlapped phase diagrams of PHMM and HD in acetone/water mixtures.
PHMM is soluble in zones a, b, and c, and micellizes in d, e, and f; HD is fully dissolved
in zones c and d, generates metastable emulsions in b and f, and demixes in a and e.
The domain f in which well-defined nanocapsules are built through the conjoint
precipitation of HD and glycopolymer is shown in green (see also Figure S6). The cloud-
occurs (f, colored green in Figure 2) is
narrow: Typically, the content of acetone
should be between 65 and 70 wt%, whereas
the final weight fraction of HD was chosen as
0.2 wt%. Dynamic light scattering (DLS) of point boundary in this diagram was established with PHMM2.
2
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Scheme 1. One-pot cross-linking and functionalization of the polymer shell.
Figure 3. A,B,C) TEM images of nanocapsules obtained by adding a solution of HD (2 mg) in acetone (0.65 g) to an aqueous polymer solution
(0.35 g of water and 1 mg of PHMM2) and cross-linking with 0.5 (A), 1 (B), and 24 equivalents (C) of IPDI with respect to the glycopolymer
chains. D) Fluorescence emission spectra of an initial solution of pyrene in acetone and of pyrene-loaded capsules. E) Aggregation of biotin-
tagged nanocapsules in the presence of avidin, as observed by DLS. F) TEM image of AuNP-functionalized nanocapsules.
droplets destabilize with time; on the other hand, in domain e,
polydisperse (and quite robust) microcapsules have been
spotted.
The versatility of the ouzo procedure also enables the
generation of more complex nanocapsule-like structures,
including the sequestration of hydrophobic molecules in the
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Angewandte
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Received: February 26, 2014
Revised: March 28, 2014
Published online: && &&, &&&&
HD core and the covalent attachment of hydrophilic mole-
cules to the nanocapsule shell[15] (Scheme 1; see also Figur-
es S7–S10). Dye-loaded nanocapsules were readily prepared
through the direct addition of hydrophobic probes (e.g.
pyrene) to the organic solution prior to the nanoprecipitation
procedure. The encapsulation of pyrene was assessed by the
fluorescence emission spectrum of the resulting dispersion.
The spectrum showed an ensemble of monomeric peaks
(between 375 and 410 nm) and an additional broad unstruc-
tured band (from 425 to 550 nm) due to excited-state-dimer
(excimer) emission, which reflects the spatial proximity of the
fluorophores in the nanocapsule cavity (Figure 3D; see
Figure S10).[16] Fluorophores or biotin were attached to the
polymer shell by incorporating an amino-functionalized
fluorescein (Fc) tag or amino-functionalized biotin in the
initial aqueous solution (see Figures S8 and S9). The addition
of these coreactants has no impact on the nanoprecipitation
and cross-linking processes. Fc-labeled and biotinylated
nanocapsules with a z-average diameter of 210 and 245 nm,
respectively, were generated. The covalent attachment of Fc
tags to the polymer shell during the nanoprecipitation process
was confirmed by a characteristic fluorescence emission at
518 nm (see Figure S9). Consistent with a successful function-
alization, biotinylated nanocapsules were proven to disrupt
4’-hydroxyazobenzene-2-carboxylic acid (HABA)-avidin
complexes owing to the favored binding of avidin to biotin
(Kd = 10À15 m versus 5.8 ꢀ 10À6 m for HABA-avidin; see also
Figure S8). Accordingly, DLS confirmed the formation of
large aggregates (967 nm) upon the addition of tetravalent
avidin to biotinylated nanocapsules (Figure 3E). One-pot
procedures with concomitant shell functionalization and core
loading were equally effective (see Figure S11). Importantly,
it was also shown that the initial incorporation of dodeca-
nethiol-functionalized gold nanoparticles (AuNPs) or iron
oxide nanoparticles within the organic phase leads to the
preparation of NP-coated nanocapsules (Figure 3F; see also
Figures S12 and S13).
Keywords: functionalization · nanocapsules ·
.
nanoprecipitation · nanostructures · phase diagrams
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different conditions from those set for the present generation of
glyconanocapsules (see the Supporting Information).
[15] Preliminary studies showed that the addition of an alkyl amine to
the aqueous phase is a simple way to consume the isocyanate
groups remaining in the shell (see Figure S7).
To emphasize the general applicability of the method, we
finally explored the preparation of nanocapsules from a differ-
ent oil, miglyol 812, and a new glycopolymer, poly(N-[2-(a-d-
mannopyranosyloxy)ethyl]methacrylamide). In analogy with
the PHMM/water/acetone/HD system, well-defined miglyol-
filled glyconanocapsules were generated, but with a smaller
diameter and lower polydispersity index (Dz = 130 nm, PDI =
0.04; see Figure S14).
In conclusion, we have demonstrated that the exploitation
of phase diagrams of different solutes solvent-shifted by the
ouzo effect enables the generation of well-defined nano-
capsules by an extremely fast and straightforward method.
The nanocapsules can be shell-cross-linked, shell-functional-
ized, and loaded with active components at the same time in
a rapid one-pot procedure. This simplified route to functional
nanocapsules is general and can be readily transposed to
other systems to design new materials of practical applic-
ability in different fields in which colloids are used.
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Angew. Chem. Int. Ed. 2014, 53, 1 – 5
These are not the final page numbers!

Angewandte
Chemie
Communications
Functional Nanostructures
X. Yan, M. Delgado, A. Fu, P. Alcouffe,
S. G. Gouin, E. Fleury, J. L. Katz,
F. Ganachaud,
J. Bernard*
&&&& — &&&&
Simple but Precise Engineering of
Functional Nanocapsules through
Nanoprecipitation
Shift N’ go! The establishment of both
polymer and oil phase diagrams facili-
tated the generation of stable nanocap-
sules through nanoprecipitation (see
picture). This simple methodology ena-
bled the preparation of shell-functional-
ized and core-loaded nanocapsules
through batch mixing.
Angew. Chem. Int. Ed. 2014, 53, 1 – 5
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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5
These are not the final page numbers!