Disclosing the nature of thermo-responsiveness of poly(N-isopropyl acrylamide)-based polymeric micelles: Aggregation or fusion?

Article abstract of DOI:10.1039/c5cc02641e

The apparent size increase of poly(N-isopropyl acrylamide) (PNIPAM)-based polymeric micelles upon heating was usually ascribed to their volume growth or aggregation in aqueous solution. Herein we designed a photo-cross-linkable PNIPAM-based copolymer and proposed another thermo-responsive behaviour-fusion, which is disclosed by transmission electron microscopy (TEM) after in situ fixing morphologies at desired temperatures.

Full text of DOI:10.1039/c5cc02641e

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Disclosing the nature of thermo-responsiveness of
poly(N-isopropyl acrylamide)-based polymeric
micelles: aggregation or fusion?†
Cite this: Chem. Commun., 2015,
51, 11198
Received 31st March 2015,
Accepted 1st June 2015
Fangyingkai Wanga and Jianzhong Du*ab
DOI: 10.1039/c5cc02641e
www.rsc.org/chemcomm
The apparent size increase of poly(N-isopropyl acrylamide) resonance (NMR),19,20 Fourier transform infrared spectroscopy
(PNIPAM)-based polymeric micelles upon heating was usually ascribed (FTIR),21–23 dynamic light scattering (DLS),24 etc. Also, some
to their volume growth or aggregation in aqueous solution. Herein we computational and simulation studies were performed to reveal
designed a photo-cross-linkable PNIPAM-based copolymer and the thermodynamic behaviour.25,26 However, these techniques
proposed another thermo-responsive behaviour – fusion, which is only provide with indirect evidence. Usually, cryogenic trans-
disclosed by transmission electron microscopy (TEM) after in situ mission electron microscopy (cryo-TEM) is a powerful tool for
fixing morphologies at desired temperatures.
visualizing the original morphologies of most soft nanostruc-
tures in solution.15 Unfortunately, one may worry about
Thermally responsive polymeric nanostructures such as micelles, whether the cryo-TEM provides direct evidence of the original
vesicles, hydrogels, etc. have attracted enormous attention over morphology of thermo-responsive polymeric nanostructures in
the past few decades.1–10 Among them, PNIPAM-based polymeric solution because the sample preparation process involves a
micelles were intensively studied. This is because PNIPAM and its significant temperature drop which may eventually induce a
derivatives have a sharp transition through lower critical solution morphological change.
temperature (LCST) and versatility in terms of copolymer archi-
tecture variation, and may be applied in a wide range of fields variation during sample preparation may be a better choice than
such as controlled drug delivery.11–13
cryo-TEM for investigating the real morphology of thermally
Therefore, conventional TEM where no obvious temperature
However, thermally responsive polymeric nanostructures still responsive soft nanostructures. The key is still how to keep their
confront some important problems. One of which is the con- original morphology in solution.27–33 Cross-linking the nanostruc-
troversial observations upon heating similar polymeric nano- ture in solution is a good choice to keep the original morphology.
particles through their transition temperatures.14 In most cases, For example, Chen et al. prepared a series of vesicles with subtle
the nature of thermal responsiveness has been regarded as the nanostructures visualized by TEM upon in situ sol–gel reactions in
volume change,15 aggregation and morphological transition.16–18 the vesicle membranes.30,31 Zhao et al. introduced photo-cross-
However, there are still questions about the volume change: (1) linking moieties in block copolymers.27,29
What is the origin of the mass when the volume of the micelle
significantly increases upon heating? (2) How is the mass (fusion of micelles) by TEM upon in situ fixing their structure in
squeezed during the volume shrinkage process upon cooling? solution. As shown in Scheme 1, a thermally responsive block
In this study, we reveal another thermally induced size increase
To answer the above questions, it is necessary to disclose the copolymer, poly(ethylene oxide)-block-poly[N-isopropyl acrylamide-
nature of thermal responsiveness of polymeric nanostructures. stat-7-(2-methacryloyloxyethoxy)-4-methylcoumarin] [(PEO-b-P(NIPAM-
Various techniques were employed, such as nuclear magnetic stat-CMA))], was directly dissolved in water to form the micelle with
a ‘fuzzy’ core at room temperature. Hydrophilic PEO chains form the
coronas of the micelle. PNIPAM is thermally responsive while PCMA is
photo-cross-linkable by UV radiation within minutes.34 The P(NIPAM-
stat-CMA) block forms the ‘fuzzy’ core, which indicates that
there is no clear boundary between the hydrophobic and hydro-
philic moieties.35,36 Upon step-by-step heating of the aqueous
solution above the LCST of PNIPAM (e.g., 45 1C), the un-cross-
linked micelles will end up in a fusional mode (route A). When
the equilibrating time is shorter (route B) or the chain mobility
a School of Materials Science and Engineering, Key Laboratory of Advanced Civil
Engineering Materials of Ministry of Education, Tongji University,
4800 Caoan Road, Shanghai, 201804, China. E-mail: jzdu@tongji.edu.cn;
Fax: +86-21-6958-4723; Tel: +86-21-6958-0239
b Shanghai Tenth People’s Hospital, Tongji University School of Medicine,
301 Middle Yanchang Road, Shanghai 200072, China
† Electronic supplementary information (ESI) available: All experimental proce-
dures, Schemes S1 and S2 and Fig. S1–S13 were listed. See DOI: 10.1039/
c5cc02641e
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Fig. 1 1H NMR spectra of PEO43-b-P(NIPAM94-stat-CMA5) micelles in
D2O at 25, 37 and 45 1C. Cpolymer = 10.0 mg mLÀ1. Samples were
equilibrated for 30 min before characterization. The signals of PNIPAM
weakened as the temperature increases. The migration of the chemical
shift to lower fields was caused by the variation of the resonance signal of
the inner standard at a higher temperature.
Scheme 1 Schematic illustration of the fusion and aggregation structures
upon heating polymeric micelles. All samples were photo-cross-linked
before the TEM study. Blue: PEO; yellow: hydrophobic PNIPAM; green:
PCMA; purple: ‘fuzzy’ core of the micelle composed of hydrophilic
PNIPAM and hydrophobic PCMA.
in the micelle core becomes less (route C), only the aggregation
of micelles occurs during the heating process.
Just to be clear, the cross-linking procedure of coumarin
moieties was utilized in this research with two different pur-
poses at two different temperatures. Cross-linking at 45 1C is
simply for fixing the morphology of nanostructures for a better
TEM study. Similarly, at 25 1C, the UV cross-linking also fixes
the original micellar structure in solution for comparing the
thermally responsive behaviour of these cross-linked micelles
with un-cross-linked micelles.
The PEO-b-P(NIPAM-stat-CMA) block copolymer was synthe-
sized through a typical reversible addition-fragmentation chain
transfer (RAFT) polymerization (see Scheme S1 in the ESI†). The
chemical structures of as-prepared PEO-based macro chain
transfer agent (macro-CTA) trithiolcarbonate, the monomer
7-(2-methacryloyloxyethoxy)-4-methylcoumarin (CMA), and the
obtained PEO43-b-P(NIPAM94-stat-CMA5) block copolymer were
Fig. 2 Size increase and corresponding interpretation of un-cross-linked
polymer micelles upon step-by-step heating to 45 1C. Cpolymer = 1.0 mg mLÀ1
.
Blue: PEO; yellow: hydrophobic PNIPAM; green: PCMA; purple: ‘fuzzy’ core of
the micelle composed of hydrophilic PNIPAM and hydrophobic PCMA.
1
confirmed by H NMR spectra in CDCl3 (Fig. S1–S3, ESI†).
At 25 1C, while PNIPAM is still water-soluble, the block (Dh) increased to 74.0 nm with a final PDI of 0.048 at 45 1C.
copolymer can be directly dissolved into deionized (DI) water During this process, the size change occurred mainly from 29 1C
at 1.0 mg mLÀ1 to form micelles of 38.9 nm (see Fig. S4, ESI†) as to 31 1C, corresponding to the LCST of PNIPAM at around 32 1C.
a result of the hydrophobic coumarin moieties and the end The corresponding size distributions and the scattered intensity
group effect.37,38 Therefore, the thermo-responsive behaviour of are presented in Fig. S5 and S6, ESI.† A heating–cooling cycle was
the PEO43-b-P(NIPAM94-stat-CMA5) block copolymer in water repeated 3 times (Fig. S7, ESI†), confirming a reversible and
1
was first evaluated by H NMR.
repeatable process.
Fig. 1 shows the 1H NMR spectra in D2O at different
To deeply understand the phenomenon in the DLS studies,
temperatures. The signals labelled ‘‘b’’ and ‘‘c’’ are associated conventional TEM was employed to investigate the morphology
with the methylene and methyl protons of the thermal respon- by in situ photo-cross-linking in solution. After step-by-step
sive PNIPAM. From 25 to 45 1C, the peak intensity of PNIPAM heating to 45 1C, the micelle solution was exposed to UV light
decreased dramatically, indicating reduced mobility and solvation for 5 min at 45 1C to fix the morphology of micelles for TEM
degree. Then, the thermal behaviour of the un-cross-linked characterization. The related photodimerization process is pre-
micelle solution was characterized by DLS. The micelle solution sented in Scheme S2, ESI.† The cross-linking degree was calcu-
was heated step-by-step from 25 1C to 45 1C with 20 min lated to be 53.6% by the variation of the characteristic peak of
equilibrating time at every 2 1C interval. This prolonged equili- coumarin at 320 nm via UV-vis spectroscopy. To prepare TEM
brating time made it possible for the thermally sensitive PNIPAM samples, the pre-heated TEM copper grids loaded with the micelle
chains to reach the equilibrium state. Fig. 2 reveals the size samples were placed in a drying oven at 45 1C to minimize the
variation during the heating process. The hydrodynamic diameter influence of temperature change on the morphology of thermally
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Table
1
Size increase after directly immersing the un-cross-linked
micelles in a water bath to quickly elevate the temperature to 45 1Ca
25 1C
45 1C
Increase in
Dia. (%)
Cini (mg mLÀ1
)
Dia. (nm) PDI
Dia. (nm) PDI
1.0
2.0
3.0
4.0
38.9
38.4
37.1
39.0
0.188 139.0
0.083 239.0
0.129 170.2
0.109 280.5
0.156 407.1
0.098 343.2
0.403 650.0
0.475 943.8
a
Samples were equilibrated for 20 min.
equilibrating time and chain mobility may be key points for the
formation of fusional structures.
To testify the above hypothesis, a series of micelle solutions
with different initial concentrations were applied with the same
step-by-step heating process. The corresponding size increases
are listed in Fig. S9, ESI.† When the concentration was as low as
0.1 mg mLÀ1, the micelle solution merely had a size change
(7.0%), indicating a concentration-dependent thermal behaviour.
The relatively low concentration diminished the aggregation and
fusion.
On the other hand, PNIPAM is known to have a fast and fully
reversible coil-to-globule transition at its LCST, which lasts
as short as hundreds of seconds.18 While in our case, the
prolonged equilibrating time is critical to the fusional struc-
ture. The micelle solution (1.0 mg mLÀ1) was then directly
immersed in a water bath at 45 1C, which was far above the
LCST of PNIPAM. The original light blue solution turned white
immediately. After equilibrating for 20 min as well, the white
solution was first studied by DLS. The results at different initial
concentrations are listed in Table 1.
As shown in Table 1, after quickly elevating the temperature
to 45 1C, the diameter of polymer micelles had a higher
proportion of increment. For example, at 1.0 mg mLÀ1, the
Dh reached 139.0 nm. In contrast, the Dh was only 74.0 nm in
the step-by-step process. This phenomenon is also highly
concentration-dependent because a higher initial concen-
tration leads to a larger final size. The TEM study (Fig. 3B
and Fig. S10, ESI†) revealed a large scale of aggregation and
clear boundaries between single micelles. The slowly tangling
and crossing process will no longer exist so that the fusional
process was hindered.
Fig. 3 TEM images of thermo-responsive polymeric micelles with differ-
ent structures upon heating. Blue: PEO; yellow: hydrophobic PNIPAM;
green: PCMA.
Although cross-linking techniques provide us with direct insight
into the morphology change and better understanding of thermo-
responsive micelles. TEM images revealed that upon heating to responsive mechanisms of nanostructures, the cross-linking tech-
45 1C, the micelles formed fusional structures consisting of two or niques themselves may inevitably have certain influences on the
several individual micelles without clear boundaries (Fig. 3A). thermal behaviour of polymers and their self-assembled nano-
Also, some unpaired micelles (ca. 40.8 Æ 4.8 nm) co-existed with structures, regardless of different cross-linking procedures. There-
the fusional structure (Fig. S8, ESI†). This phenomenon is unique fore, we introduced the cross-linking technique at the beginning
in the PNIPAM-based micelles and it is interpreted that the at 25 1C to compare different thermal behaviours between the
prolonged equilibrating time below LCST accelerated the move- un-cross-linked micelles and cross-linked micelles caused by dif-
ment and collision of individual micelles, thus resulting in more ferent chain mobilities.
chance to encounter, tangle and cross mutually. When the
The directly dissolved micelle solution was placed under UV
temperature was above the LCST, tangled hydrophilic chains spotlight to cross-link for 3 min. The Dh after photo-cross-
became water-insoluble so as to create a hydrophobic environ- linking was 32.9 nm (Fig. S11, ESI†) and the cross-linking
ment nearby. Under these circumstances, solution concentration, degree of the micelle was 42.5% (up to 56.3% in 10 min, see
11200 | Chem. Commun., 2015, 51, 11198--11201
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J.D. is supported by National Natural Science Foundation of
China (21174107 and 21374080), Shanghai 1000 Plan, the
Eastern Scholar program and the open fund for characteriza-
tion at Tongji University (0002014118).
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