Synthesis and self-assembly of nonamphiphilic hyperbranched polyoximes

Article abstract of DOI:10.1039/c2sm26124c

Nonamphiphilic hyperbranched polyoximes (HPOXs) were successfully synthesized by the polycondensation of trialdehyde and bis-aminooxy monomers with different molar feeding ratios. Various characterization techniques, such as 1D and 2D nuclear magnetic res

Full text of DOI:10.1039/c2sm26124c

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PAPER
Synthesis and self-assembly of nonamphiphilic hyperbranched polyoximes†
a
a
a
a
a
Yue Jin, Liang Song, Dali Wang, Feng Qiu, Deyue Yan, Bangshang Zhu and Xinyuan Zhu
b
ab
*
Received 16th May 2012, Accepted 19th June 2012
DOI: 10.1039/c2sm26124c
Nonamphiphilic hyperbranched polyoximes (HPOXs) were successfully synthesized by the
polycondensation of trialdehyde and bis-aminooxy monomers with different molar feeding ratios.
Various characterization techniques, such as 1D and 2D nuclear magnetic resonance (NMR), Fourier
transform infrared spectroscopy (FTIR), and multi-detector gel permeation chromatography (GPC)
were used to identify the highly branched structure of the HPOXs. Despite there being no amphiphilic
block segments, HPOXs with a torispherical structure could self-assemble into nanoparticles in a mixed
solution of dimethyl sulfoxide and H2O. Besides, the modulation of the degree of branching (DB) and
the terminal groups resulted in the appearance of spherical and bowl-shaped morphologies due to the
change of the intra- and inter-molecular interactions. Accordingly, dynamic light scattering (DLS),
transmission electron microscopy (TEM), atomic force microscopy (AFM), scanning electron
microscopy (SEM), fluorescence spectroscopy (FL) together with ultraviolet and visible spectrometery
(UV-vis) were employed to unravel the tentative mechanism of the formation of the HPOX self-
assemblies. Moreover, dynamic oxime linkages and hydrogen bonds endowed the HPOX nanoparticles
with pH and thermal dual responsiveness, which was confirmed by TEM measurements. HPOXs are
developed to offer a novel pathway for the design of nonamphiphilic self-assemblies with dual
responsiveness.
As a matter of fact, natural assemblies without a well-defined
separation of the hydrophilic–hydrophobic blocks, such as
protein folding, are ubiquitous. The subunits or peptide chains of
sophisticated proteins intertwine into coils and further converge
into globular micelles, and the self-assembled structures are
closely related to their functions. It’s clear that noncovalent
interactions like multiple hydrogen bonding interactions play a
crucial role in the physical structures and chemical functions of
natural aggregates, the manipulation of which allows access to
excellent bio-inspired materials with versatile building blocks.3–11
Similar to natural proteins, nonamphiphilic dendritic polymers
are blessed with intrinsic torispherical conformation and
numerous functional groups, which provide sufficient sites for
supramolecular interactions and self-assembly. Furthermore, the
adjustment of branched architecture and functional terminals is
capable of effectively altering the supramolecular interactions.
Hence, it can be imagined that by means of mimicking the struc-
tures and interactions of natural biomacromolecules, the self-
assembly of nonamphiphilic synthetic polymers might be realized.
In this work, a three-dimensional hyperbranched polyoxime
(HPOX) incorporating dynamic oxime bonds, numerous amide
groups and aminooxy terminals, was prepared by the poly-
condensation of bis-aminooxy and trialdehyde.12–14 As expected,
the novel HPOXs without defined amphiphilic segments could
self-assemble into nanoparticles, attributed to the inter- and
intra-molecular multiple noncovalent interactions.15,16 In
particular, changing the surface functional groups and branched
Introduction
The solvophilic–solvophobic interaction of amphiphilic poly-
meric blocks can fabricate common nanoparticles with typical
core–shell structure or other morphologies. From the traditional
perspective of self-assembly, the distinct amphiphilic structure is
indispensable for the formation of polymeric nanoparticles. For
nonamphiphilic polymers, it is difficult to provide enough inter-
and intra-molecular interactions to drive the formation of
nanoparticles. Therefore, the design of nonamphiphilic struc-
tures that can spontaneously aggregate into micelles has greatly
challenged the common self-assembly principle of synthetic
macromolecules.1,2
aSchool of Chemistry and Chemical Engineering, State Key Laboratory of
Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan
Road, Shanghai 200240, People’s Republic of China
bInstrumental Analysis Center, Shanghai Jiao Tong University, 800
Dongchuan Road, Shanghai 200240, People’s Republic of China. E-mail:
xyzhu@sjtu.edu.cn; Fax: +86-21-34205722; Tel: +86-21-34205699
† Electronic supplementary information (ESI) available: mass spectrum
of TBA. The dendritic, linear and terminal structural units of the
HPOXs are listed and the DB of the HPOXs was calculated by
quantitative 13C NMR spectroscopy. The TGA and DSC curves of the
HPOXs with different molar feeding ratios of trialdehyde to
bis-aminooxy monomers. An AFM image of HPOX 3. 1H NMR
spectra of HPOX 4 in a mixture of DMSO-d6 and D2O and the
degradability of the oxime bonds after adding HCl for 4 h. See DOI:
10.1039/c2sm26124c
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architecture of the HPOX led to the morphological trans-
formation from spherical to bowl-shaped micelles.17–19 Both
oxime linkages and hydrogen bonds endowed the HPOXs with
stimuli responsiveness. The self-assembly of nonamphiphilic
dendritic polymers provides a new strategy for designing stimuli-
responsive nanostructures with a vast potential for further
applications.
multi-detector calibration of the optical constant was carried out
with a poly(methyl methacrylate) (PMMA) standard (Mn
¼
750 000 g molꢁ1, PDI ¼ 1.12). The molecular weight and the
polydispersity index (PDI) were calculated using OmniSEC 3.1
software.
Differential scanning calorimetry (DSC). DSC was performed
on a TA Q2000 series in a nitrogen atmosphere. Indium and zinc
standards were used for temperature and enthalpy calibration.
First, the samples were heated from room temperature to 200 ꢀC,
held at this temperature for 3 min to remove the thermal history
and then slowly cooled to ꢁ50 ꢀC at 10 ꢀC minꢁ1. Subsequentꢀly,
Experimental section
Materials
O,O0-1,3-Propanediylbishydroxylamine (PBH, Aldrich, 99%),
terephthalaldehydic acid (TPA, TCI, >98%), N-hydroxy-
succinimide (NHS, Acros, >98%), 1,3-dicyclohexylcarbodiimide
(DCC, Aldrich, 99%), tris(2-aminoethyl) amine (TAA, Alfa
Aesar, 97%) and 1,6-diphenyl-1,3,5-hexatriene (DPH, 98%) were
used as received without further purification. N,N-Dime-
thylformamide (DMF) and dimethyl sulfoxide (DMSO) were
used after purification by distillation under vacuum and dried
with calcium chloride (CaCl2) and calcium hydride (CaH2),
respectively. Dichloromethane, chloroform and isopropanol
were refluxed with CaH2, and then distilled prior to use. All other
reagents and solvents of analytical grade were purchased from
Sinopharm Chemical Reagent Co., Ltd and used as received
unless otherwise mentioned.
ꢀ
ꢀ
the samples were heated again from ꢁ50 C to 200 C at 10 C
minꢁ1 to determine their glass transition temperatures (Tg).
Thermal gravimetric analysis (TGA). TGA measurements were
performed on
a Perkin-Elmer TGA-7 thermo-gravimetric
analyzer to investigate the thermal stability of all the samples in a
ꢁ1
ꢀ
ꢀ
nitrogen atmosphere from 40 C to 700 C at 20 ꢀC min
.
Dynamic light scattering (DLS). DLS measurements were
performed with a Malvern Zetasizer Nano S instrument (Mal-
vern Instruments Ltd) equipped with a 4.0 mW He–Ne laser
operating at l ¼ 633 nm. All samples (1 mg mLꢁ1) were
measured at a scattering angle of 173ꢀ in aqueous solution at
ꢀ
room temperature (25 C).
Characterization
Transmission electron microscopy (TEM). TEM studies were
performed with a JEOL JEM-100CX-II instrument at a voltage
of 200 kV. Samples were prepared by drop-casting micelle
solutions onto carbon-coated copper grids, and then air-drying
at room temperature before measurement.
Nuclear magnetic resonance (NMR) spectroscopy. H and 13C
1
NMR analyses were recorded on a Varian Mercury Plus 400
MHz spectrometer with deuterated dimethyl sulfoxide (DMSO-
d6) and deuterated chloroform (CDCl3) as solvents at 20 ꢀC.
Tetramethylsilane (TMS) was used as the internal reference. 13C,
1H-heteronuclear singular quantum correlation (HSQC) and
Atomic force microscopy (AFM). The morphology was visu-
alized using an atomic force microscope (AFM) with a tapping
mode and a Nanoscope IIIa controller (Bruker, Veeco/DI). Tip
information: radius <10 nm, cantilever length 90 ꢂ 5 mm; width
40 ꢂ 3 mm; thickness 2.0 ꢂ 0.5 mm, resonant frequency 330 kHz,
1
13C, H-heteronuclear multiple bond correlation (HMBC) were
carried out using a Bruker Avance III 400 spectrometer with
DMSO-d6 as the solvent. Quantitative 13C NMR spectra were
1
measured by the method of inverse gated H decoupling.
force constant 48 N mꢁ1
.
Mass spectroscopy (MS). The purity was analyzed by qua-
tropde-time of flight mass spectrometer (Q-TOF-MS) measure-
ments, which were performed on a Waters-ACQUITYTM
UPLC & Q-TOF-MS Premier with acetonitrile as the solvent.
Scanning electron microscopy (SEM). The morphology was
visualized using a scanning electron microscope after gold
sputtering treatment (FEI Nova NanoSEM 230 LV UHR
FE-SEM OxFORD INCA X-Max 80 SDD EDS).
Fourier transform infrared (FTIR) spectroscopy. FTIR spectra
were measured by the KBr sample holder method on a Perkin
Elmer Paragon 1000 instrument in the range 3600–400 cmꢁ1
Fluorescence spectroscopy (FL). The fluorescence spectra were
measured on a QM/TM/IM steady-state and time-resolved
fluorescence spectrofluorometer in the range 375–700 nm.
(Excitation wavelength lex ¼ 360 nm).
.
Gel permeation chromatography (GPC). The molecular
weights and the Mark–Houwink–Sakurada parameters were
determined on a Viscotek GPCMax VE2001 instrument with a
quadruple-detector (differential refractive index (RI), UV-vis,
differential viscometer (intrinsic viscosity, IV) and right-angle
light scattering (RALS)). The GPC instrument was equipped
with three Jordi polydivinylbenzene columns (Jordi FLP, Bur-
Synthesis of N-succinimidyl 4-formylbenzoate20
TPA (3 g, 22.4 mmol) and NHS (2.3 g, 20 mmol) were dissolved
in a mixture of DMF (10 mL) and dichloromethane (27 mL)
under an argon atmosphere. The solution was cooled to around
0 ꢀC in an ice–salt bath, followed by gradually adding 4 mL of a
dichloromethane solution of DCC (4.1 g, 20.0 mmol) in an
airtight reaction vessel. After removing the ice–salt bath, the
vessel was continuously stirred at room temperature overnight.
ꢀ
lingham, MA) with pore sizes of 100 000, 10 000, and 1000 A,
ꢀ
respectively. The column temperature was set to 40 C. HPLC-
grade DMF (n ¼ 1.430) with LiBr at a concentration of 0.05 M
was used as the mobile phase at a flow rate of 1.0 mL minꢁ1. The
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The precipitated N,N0-dicyclohexylurea (DCU) was removed by
filtration. Dichloromethane was removed from the filtrate by
rotatory evaporation and the remaining solution was frozen in
liquid nitrogen and lyophilized to remove the DMF solvent. The
dry residue was dissolved in 135 mL of isopropanol under reflux.
The hot solution was immediately filtered and then cooled to
room temperature. Subsequently, the precipitate was collected by
filtration and dried under vacuum at 45 ꢀC for 24 h. A white
product was obtained. Yield: 2.5 g, 50%.
1H NMR (400 MHz, CDCl3, 298 K, ppm) d: 2.92 (4H, s,
–OCH2CH2CO–), 8.02 (2H, d, J ¼ 7.04), 8.30 (2H, d, J ¼ 7.04),
10.13 (1H, s, –CHO). 13C NMR (400 MHz, CDCl3, 298 K, ppm)
d: 25.86, 129.90, 131.47, 140.63, 161.12, 169.14, 191.77.
in Table 1 and Table S1 in the ESI†) is as follows: PBH (59.68
mg, 1/3 mmol) was dissolved in anhydrous DMSO (5 mL) in a
flask, and a DMSO solution (10 mL) of TBA (180.86 mg, 1/3
mmol) was added dropwise (the molar ratio of the two mono-
mers was set at 1.0). The flask was consecutively evacuated and
ꢀ
refilled with N2 three times and then submerged in a 37 C oil
bath for 48 h. The light yellow reaction solution was transferred
into a dialysis bag (MWCO ¼ 3.5 kDa), which was enclosed in
2 L deionized water and dialyzed with renewed water for 48 h.
After dialysis, the dialysis solution was frozen and lyophilized to
a constant weight by a freeze-dryer system (Martin Christ, a1-4,
ꢀ
Germany) at ꢁ56 C for 48 h. The HPOXs with different DBs
were obtained.
1H NMR (400 MHz, DMSO-d6, 298 K, ppm) d: 2.07 (2H,
br, –CH2CH2ONC–), 2.70 (2H, br, –NCH2–), 3.32 (2H,
br, –CH2NH–), 4.24 (2H, br, –CH2ONC–), 7.32 and 7.56
(2H, br, Ar), 7.48 and 7.75 (2H, br, Ar), 7.86 (2H, br, Ar), 7.90
(2H, br, Ar), 8.14 (2H, br, Ar), 8.25 (1H, br, –CH]NO–), 8.38
(1H, br, –CONH–), 8.55 (2H, br, –ONH2), 10.00 (1H, br, CHO).
13C NMR (400 MHz, DMSO-d6, 298 K, ppm) d: 29.3, 38.2, 39.5–
40.8, 53.6, 71.2, 127.2, 128.2, 128.5, 130.0, 135.0, 135.7, 138.3,
140.0, 148.8, 166.4, 193.4.
Synthesis of tris(2-(4-formylbenzamide) ethyl) amine21–24
TAA (243.7 mg, 5/3 mmol) was completely dissolved in 25 mL
DMSO. The reaction at room temperature was initiated by
adding another 25 mL DMSO solution of N-succinimidyl
4-formylbenzoate (1.236 g, 5 mmol). After 2 h, the vessel was
filled with 100 mL H2O. After another 2 h, the reaction solution
was extracted with 3 ꢃ 40 mL chloroform. The mixed organic
phase was washed with 50 mL water and dried by anhydrous
magnesium sulfate (MgSO4). After precipitation, the solvent was
removed by rotary evaporation. The pale yellow solid tris(2-(4-
formylbenzamide) ethyl) amine (TBA, N(CH2CH2NHCO–
C6H4–p–CHO)3) was sonicꢀated in 250 mL water, filtered and
dried under vacuum at 45 C for 48 h to afford a slightly off-
white powder. Yield: 0.57 g, 64%.
Preparation of the HPOX micellar solution
Firstly, ꢄ4 mg of the synthetic HPOXs were completely dis-
solved in 3.2 mL DMSO. Next, 0.8 mL deionized water was
added dropwise under continuous stirring at room temperature,
making a final HPOX concentration of 1 mg mLꢁ1. The resulting
micellar solution was prepared to observe the morphology of the
HPOXs. The appearance of turbidity in the solution indicated
the formation of the aggregates.
1H NMR (400 MHz, DMSO-d6, 298 K, ppm) d: 2.73 (2H,
t, –NCH2–), 3.39 (2H, m, –CH2NH–), 7.85 (2H, d, J ¼ 8.61), 7.91
(2H, d, J ¼ 8.61), 8.54 (1H, t, –CH2NH–), 10.01 (1H, s, CHO).
13C NMR (400 MHz, DMSO-d6, 298 K, ppm) d: 37.7, 53.7,
128.0, 130.0, 140.3, 167.3, 191.0.
Critical aggregation concentrations of self-assembled HPOX
aggregates
Ulta performance liquid chromatography (UPLC) & Q-TOF-
MS of TBA (Fig. S1†): calculated for [M + H]+: 543.22, found
m/z: 543.2244 [M + H]+.
Here, DPH was used as the UV-vis probe to investigate the
critical aggregation concentration (CAC) of the HPOX self-
assemblies. Initially, the appropriate concentration of the HPOX
solution was prepared and diluted to various pre-set concentra-
tions (from 0.1 mg mLꢁ1 to 5.0 ꢃ 10ꢁ5 mg mLꢁ1) by DMSO
solvent mixed with 20% H2O. Secondly, a 0.5 mmol Lꢁ1 meth-
anol solution of DPH was added into the serial solutions, making
the DPH concentration at a constant of 5.0 ꢃ 10ꢁ6 mol Lꢁ1. The
set wavelength absorbance of 313 nm of all the solutions was
recorded on a Perkin Elmer Lambda 20 UV-vis spectrometer.
The points and the fitting lines were obtained and the horizontal
ordinates of the turning points gave the CAC values of the
different HPOX samples.
Synthesis of HPOXs with different molar ratios of monomers
An oximization polymerization procedure (where the molar
feeding ratios of bis-aminooxy to trialdehyde vary from 0.8 to 1.5
Table 1 GPC data and thermal properties
Entry
HPOX 1
HPOX 2
HPOX 3
HPOX 4
TBA : PBHa
1 : 0.8
6.2
1.7
0.278
0.139
0.51
339.9
31.5
81.4
1 : 1.0
12.3
3.2
0.282
0.229
0.38
336.8
30.7
87.1
1 : 1.2
31.7
4.2
0.289
0.223
0.32
333.6
22.5
91.5
1 : 1.5
49.8
4.4
0.306
0.191
0.23
329.1
25.8
103.2
Mw (ꢃ103)
b
PDIb (Mw/Mn)
ab
dn/dcb
DBc
Thermo- and pH-responsiveness of the HPOX nanoparticles
d
Td-10% (ꢀC)
Wresidue (%)
d
For thermo-responsiveness, the HPOX 3 micellar solution was
heated to 80 ꢀC and then cooled to room temperature. The
alteration of the turbidity was observed. The reversible diameter
changes were monitored by DLS techniques between 25 and 85
ꢀC. The equilibrium time at different temperatures was kept
above 30 min. For pH-responsiveness, aqueous dilutions of tri-
fluoroacetic acid (TFA) and triethylamine (TEA) were added
into the HPOX 3 micellar solution, respectively. Subsequently,
e
Tg (ꢀC)
a
b
The molar ratio of TBA to PBH. Molecular weights, polydispersity
c
and Mark–Houwink a determined by multi-detector GPC. Degree of
branching determined by quantitative 13C NMR spectroscopy. The
temperature at 10% weight loss and the residual weight percentage at
d
e
700 ꢀC determined by TGA. The glass transition temperature
determined by DSC.
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the pH values were modulated at 4 and 10, respectively. After
stirring for 3 h, observation of the turbidity of the aggregate
solutions was made and TEM micrographs were taken to
investigate the morphology of the aggregates at different pH
values. Besides, the reversible diameter changes were monitored
by DLS techniques in the different pH environments (pH 4, 10
and 7). The equilibrium time at the different pH values was kept
above 30 min.
Results and discussion
Synthesis and characterization of the HPOXs
The synthetic route of the nonamphiphilic hyperbranched pol-
yoximes (HPOXs) is illustrated in Scheme 1. HPOXs were
prepared through the A2 + B3 oxime coupling reaction of the bis-
aminooxy and tri-aldehyde monomers. Benefiting from the
highly branched configuration, HPOXs possess a considerable
number of end groups. By altering the molar feeding ratios of the
A2 to B3 monomers, the terminal groups of the HPOXs can be
readily converted from aldehydes to aminooxy functional
groups. Both aldehyde and aminooxy groups are favorable for
the effective adjustment of intra- and inter-molecular interac-
tions and further modification, such as chemoselective ligations
as robust biomolecular hooks.25–31 To confirm the existence of
the highly branched structure of the HPOXs, 1H NMR, 13C
NMR, 13C, 1H-HSQC, and 13C, 1H-HMBC NMR techniques
Fig. 1 1H NMR spectra of the HPOXs with different molar feeding
ratios of trialdehyde to bis-aminooxy monomers.
were exploited. Fig. 1 shows the variation of the 1H NMR
spectra of the HPOX series. The proton signal at d ¼ 10.0 ppm,
assigned to the aldehyde end groups, is obviously impaired by
increasing the amount of the bis-aminooxy monomer. The
aldehyde signal disappears when the aminooxy/aldehyde ratio
arrives at 1.5, confirming the complete reaction of the aldehyde
terminals and thus the formation of the highly branched product.
The degree of branching (DB) is an important topological
parameter for hyperbranched polymers. Generally speaking, the
DB of hyperbranched polymers is determined by means of NMR
analysis of the different structural fractions. The similar chemical
environments among the dendritic units (D), the terminal units
(T) and the linear units (L) in the dendritic structure of the
HPOXs give rise to the overlapping signals in the 1H NMR
spectra between 7.2 and 8.6 ppm ascribed to the phenyl, oxime,
amide and aminooxy protons, respectively. Accordingly, 2D
13C,1H-HMBC and 13C,1H-HSQC spectra are employed to
distinguish the overlapping peaks, and the assignments are given
in Fig. 2. To calculate the DB, the D, L and T units of the
HPOXs are listed in Fig. S2 in the ESI.† Correspondingly, the
DB of the samples was determined according to the following
equation:32
DB ¼ (D + T)/(D + T + L)
For the HPOXs, the DB was calculated from quantitative 13C
NMR analysis. As shown in Table 1, the DB of the HPOX
samples range from 0.23 to 0.51, substantiating the highly
branched architecture.
The FTIR spectra in Fig. 3 indicate that the absorption band
at 1707 cmꢁ1 from the C]O stretching of the aldehyde terminals
decreases with the increasing amount of bis-aminooxy and finally
disappears. Simultaneously, the N–H absorption band at around
3420 cmꢁ1 due to the asymmetrical stretching vibration of the
aminooxy group is gradually separated from the broad O–H
absorption band and becomes evident. The FTIR spectra
demonstrate the successful control of the terminal groups with
altering the monomer feeding ratios. Moreover, the FTIR
Scheme 1 Schematic representation of the synthetic route of the HPOXs
and the tentative mechanism of the HPOX self-assembling behavior.
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Fig. 3 The FTIR spectra of the HPOXs with different molar feeding
ratios of trialdehyde to bis-aminooxy monomers.
polymerization. In contrast, the molecular weight declines when
the ratios deviate from 1. Herein, the ratio of HPOX 4 is 1, so it is
reasonable that its molecular weight is the largest. The Mark–
Houwink parameter a is frequently used to reflect the branched
structure of polymers based on the Mark–Houwink equation
([h] ¼ K ꢃ Mah). From the Mark–Houwink fitting lines of the
HPOX series, the parameter a lies between 0.27 and 0.31, con-
firming the formation of the hyperbranched structure. Addi-
tionally, the DSC and TGA curves are listed in Fig. S3 and S4 of
the ESI.† The thermal stability of the HPOXs was investigated
by TGA, wherein the Wresidue data reveals the high thermal
stability of HPOX with above 25% of the residue remaining even
at the high temperature of 700 ꢀC. Based on the chemical
structure of the HPOXs, it can be found that they belong to a
kind of polyamide. Therefore, the HPOXs have a good thermal
stability, especially due to the existence of the aromatic groups.
After thermal degradation, the residues were observed to be
black with very poor solubility in common organic solvents.
According to the literature,33–35 the residues may include organic
carbide and certain aromatic derivatives under the inert atmo-
sphere. The detailed GPC data together with the thermal prop-
erties of the HPOXs are summarized in Table 1. The solubility of
the HPOXs was also investigated and all the HPOX samples
manifested excellent solubility in common organic solvents, such
as ethanol, DMSO and DMF. In terms of the traditional
concept, the nonamphiphilic HPOXs weren’t supposed to
aggregate. Surprisingly, it was found that the synthetic HPOXs
self-assembled into nanoparticles in a mixture of DMSO and
H2O. Therefore, a series of experiments were carried out to
elucidate this phenomenon, and the tentative mechanism for the
self-assembling behavior of the HPOXs in the mixed solution
was proposed.
Fig. 2 The 2D NMR spectra of HPOX 3: (A) the 13C,1H-HMBC
spectrum, and (B) the 13C,1H-HSQC spectrum.
spectra reveal the additional chemical structure information of
the HPOX samples, which are consistent with the 1H NMR
analysis.
Molecular weight, thermal stability and solubility
The number-average molecular weight (Mn), polydispersity
(Mw/Mn), viscosity-average molecular weight (Mh) and Mark–
Houwink parameter (a) were measured by the multi-detector
GPC technique. It should be mentioned that the molecular
weight determination of hyperbranched polymers is still a
problem to be solved. Given that the hydrodynamic volume of a
hyperbranched polymer is smaller than that of the corresponding
linear polymer with a similar molecular weight, the actual
molecular weights of the HPOXs are supposed to be larger than
the results from the multi-detector GPC test. As shown in Table
1, the molecular weights of the HPOXs increase with the amount
of bis-aminooxy monomer, because the largest molecular weight
can be obtained when the molar feeding ratio between the
reactive aminooxy and aldehyde groups arrives at 1 during the
Investigation of HPOX self-assembly
To confirm the formation of the HPOX self-assemblies, UV-vis
spectroscopy in conjugation with DLS, TEM, AFM, SEM and
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fluorescence spectrometry was comprehensively exploited to
prove the formation of the HPOX nanoparticles, illustrating the
tentative mechanism for the self-assembling behavior of the
HPOXs.
The CAC values of all the samples (HPOX 1, 2, 3, and 4) were
measured by the UV-vis technique utilizing DPH as a probe. As
shown in Fig. 4, the absorbance of DPH is near to zero while the
concentration is below the CAC value, whereas the absorbance
intensity of DPH is enhanced exponentially with concentration
when the solvophobic DPH probe is packed in the micelles.
Subsequently, the two fitting lines reflecting two states intersect
at the turning point, the horizontal ordinate of which determines
the CAC value. The CAC values of the HPOX samples are
approximately close to each other in the same order of magni-
tude, approximately 10ꢁ2 mg mLꢁ1, exhibiting the opposite
effect of the DB and hydrophilic–hydrophobic ratios of the
HPOXs in the same mixed solution. The CAC determination
verifies the formation of the HPOX micelles in the mixed media.
The hydrodynamic diameter and size distribution of the
HPOX nanoparticles at the concentration of 1.0 mg mLꢁ1 in the
mixed DMSO–H2O solvent were determined by the DLS tech-
nique, and the results are given in Fig. 5. With increasing
amounts of the bis-aminooxy monomer, the average hydrody-
namic diameters of the HPOX micelles increase from 282 nm to
582 nm. The polydispersity indexes (PDIs) of HPOX 1, 2, 3 and 4
Fig. 5 DLS curves of the hydrodynamic diameters of the different
HPOXs at a concentration of 1.0 mg mLꢁ1
.
nanoparticles are observed in Fig. 7(b) and (d), and S5 of the
ESI,† confirming the capability of controlling the morphologies
of the nonamphiphilic micelles by changing the important
structural parameters of the highly branched polyoximes. All the
morphological data are listed in Table 2.
According to the previous report,18,19 it was suggested that the
formation of the bowl-shaped structure was kinetically
controlled. At the beginning of the water addition to the HPOX–
DMSO solution, the initial structure of the self-assembly is an
apparently large sphere benefiting from the strong intermolec-
ular interaction of the HPOX 4 chains, the viscosity of which is
low. Therefore, the solvent molecules diffuse rapidly with the
high chain mobility. Upon further addition of water, DMSO
continues to be extracted through the surface of the sphere,
are low, suggesting
nanoparticles.
a narrow size distribution of these
The morphologies of the as-prepared HPOX self-assemblies
were observed by TEM, and the images are shown in Fig. 6.
Consistent with the results of DLS, the TEM images indicate the
increasing size of the HPOX 1, 2, and 3 nanoparticles with the
increasing amount of the bis-aminooxy monomer. The sizes are
smaller than those measured with the DLS technique, as TEM
and DLS show different morphologies in the solid and swollen
states, respectively. As illustrated in Fig. 6, the shapes of HPOX
1, 2 and 3 are different from that of HPOX 4. Correspondingly,
the AFM and SEM images of HPOX 3 and HPOX 4 were
measured to verify the formation of the HPOX nanoparticles
with different morphologies. Both spherical and bowl-shaped
Fig. 4 The relationship of the absorbance intensity of DPH as a function
Fig. 6 TEM images of (a) HPOX 1, (b) HPOX 2, (c) HPOX 3, and (d)
of the concentration with the different HPOX samples.
HPOX 4. (Scale bar: 500 nm).
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different D2O volumes at 300 K were measured to demonstrate
the effect of the water content on the self-assembling behavior of
the nonamphiphilic nanoparticles in the mixed medium.
Accordingly, the peak of oxime bonds disappears with the
addition of water, indicating the inclination of the oxime bonds
inside the nanoparticles due to the hydrophobicity. The intro-
duction of H2O transforms the conformation of the HPOXs in
the mixed solution. It could be inferred that the intra- and inter-
molecular noncovalent interactions mainly stem from oxime
bonds, aminooxy, amide and phenyl groups, hydrogen bonding
interactions, p–p stacking interactions and the exchange of R
groups of the oxime bonds (C]N–OR) with the aminooxy
terminals, which may exert a great influence on the interwoven
self-assembling structures.37–39
To explore the tentative mechanism, fluorescence emission
spectroscopy was utilized to testify whether the p–p stacking
interaction contributed to the self-assembling behavior. Fig. 8
shows that the fluorescence emission spectra of the HPOX 2
aggregates in the mixture apparently distinguish from that of the
spectrum of HPOX 2 in pure DMSO, exhibiting an intense
maximum at about 465 nm and a shoulder at 538 nm under an
excitation wavelength of 360 nm. By comparison with the fluo-
rescence spectrum before self-assembly, the micellar solutions of
HPOX 2 with different volume ratios of DMSO to H2O reveal
the relatively higher emission intensity, demonstrating that the
p–p stacking of phenyl rings doesn’t emerge, otherwise it would
result in luminescence quenching, which can greatly diminish the
fluorescence emission intensity.40 Hence, it can be inferred that
the p–p stacking interaction may have little relationship with the
interactions for aggregation. In other words, multiple hydrogen
bonding interactions among the amide, oxime and aminooxy
groups play the central role in HPOX micellization, which may
cause the steric hindrance of the p–p stacking of the aromatic
groups. As a result, the fluorescence emission doesn’t diminish
after self-assembly, as shown in Fig. 8. By adjusting the molec-
ular conformation, the nonamphiphilic HPOXs with a tori-
spherical structure can self-assemble into nanoparticles with
spherical and bowl-shaped morphologies. The conformation
Fig. 7 HPOX 3 spherical self-assemblies: (a) AFM image (10 mm), (b)
SEM image, and HPOX 4 bowl-shaped self-assemblies: (c) AFM image
(10 mm), (d) SEM image.
Table 2 Morphological properties of HPOX aggregates (1.0 mg mLꢁ1
Diametera (nm) PDIa TEMb (nm) CACc (mg mLꢁ1
)
)
Entry
HPOX 1 282
HPOX 2 301
HPOX 3 498
HPOX 4 582
0.166 ꢄ150
0.033 ꢄ250
0.174 ꢄ480
0.235 ꢄ550
0.015
0.010
0.014
0.017
a
b
The average diameters and PDI determined by DLS. The average
diameters determined by TEM. The CAC values determined by the
UV-vis absorbance intensity of DPH.
c
leading to the high viscosity of the core. The low mobility of the
HPOX chains can result in the external shell hardening. Alter-
natively, a liquid–liquid phase separation may occur. Subse-
quently, bubbles filled with the solvent-rich phase can form. The
DMSO–water-filled bubbles are able to coalesce into a single
bubble due to the good solubility and low viscosity of the highly
branched architecture of HPOX 4, the driving force for which is
the interfacial energy. The single bubble breaks through the
surface but can’t establish a spherical shape. Correspondingly, a
bowl-shaped nanostructure can be formed. However, HPOX 1,
2, 3 don’t form bowl-shaped nanoparticles, because the added
viscosity-control mechanism plays an important role as well.36 At
the same water content, the added viscosity control of HPOX 4 is
probably provided by more hydrogen bond interactions among
the aminooxy groups at the periphery and the amide groups
along the backbone in comparison with the samples of HPOX 3,
2 and 1 with decreasing amounts of aminooxy end groups.
Therefore, the adjustment of the branched architecture can
control the morphology of HPOX self-assemblies.
DMSO is the preferential solvent for HPOX, but H2O
1
provides poor solvation. In Fig. S6 of the ESI,† the H NMR
Fig. 8 Fluorescence emission spectra of (a) HPOX 2 in pure DMSO
solvent, and HPOX 2 nanoparticles in DMSO–H2O with different
volume ratios of (b) 4 : 1, (c) 2 : 3, and (d) 1 : 4 (lex ¼ 360 nm).
spectra of HPOX 4 in the mixture of DMSO-d6 and D2O with
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alteration is driven by the intra- and inter-molecular interactions
from the changes in the structural parameters of the HPOXs,
which may mimic the route of the natural assemblies.
Thermal and pH dual responsiveness of HPOX self-assembly
The photographs in Fig. 9 present the apparent transformation
of turbidity in a heating–cooling cycle for the observation of
HPOX self-assemblies. When the micellar solution is gradually
heated to 85 ꢀC, the turbid mixed solution evidently becomes
optically clear, indicating the dissociation of the HPOX aggre-
gates with increasing temperature. Conversely, when cooled back
to room temperature, it spontaneously returns to the former
turbidity, suggesting the re-assembly of HPOX after disas-
sembly. The reversible thermal-responsive property of the HPOX
self-assemblies was further confirmed by the DLS technique. The
diameter ratio between 25 and 85 ꢀC is denoted by the formula of
size/498. The result is in harmony with the proposed mechanism,
which derives from hydrogen bonds and reversible oxime bonds.
The high temperature accelerates the breaking of the weak
linkages of the hydrogen bonds and oxime bonds.
The photos in Fig. 10 also exhibit the obvious discrepancy in
turbidity of HPOX micellar solutions with varying pH values (4,
7 and 10), reflecting pH responsiveness. It should be mentioned
that the hydrolysis of labile oxime bonds is catalyzed by general
acids and bases. Both the dynamic oxime bonds and hydrogen
bonds of the HPOXs are subjected more to the acidic conditions
(pH 4) than the alkaline medium (pH 10).41–43 At pH 4, the acidic
conditions not only facilitate the destruction of the hydrogen
Fig. 10 (a) Photographs with altering pH, (b-1, b-2 and b-3) TEM
images of the HPOX 3 self-assemblies (scale bars: 200 nm and 500 nm)
with altering pH, and (c) their reversible diameter changes at 25 ꢀC with
altering pH (4, 10 and 7). (Diameter ratio: size/498).
bonding interactions by protonation, but also accelerate the
hydrolysis of the conjugated oxime bonds. The breaking of
both the hydrogen bonds and oxime bonds results in the
complete disappearance of the turbidity of the HPOX micelles.
The environment at pH 10 only catalyzes the reversible
decomposition of part of the oxime bonds. Consequently, the
breaking of these oxime bonds leads to the shrinking of the
HPOX micelles into smaller ones, thus making the turbidity
decrease. The TEM images in Fig. 10 provide potent support
for the morphological changes with the different pH values (4, 7
and 10). As observed, the disruption of the HPOX micelles in
the TEM micrographs occurs at pH 4, while the size of the
nanoparticles diminishes at pH 10, the results of which are
consistent with the alteration of the turbidity in the photo.
Likewise, the reversible pH-responsive property of the HPOX
self-assemblies was further confirmed by the DLS technique.
The diameter ratio with altering pH values is denoted by the
formula of size/498. The reversibility was monitored and
confirmed by DLS for 4 cycles. Therefore, both the dynamic
oxime covalent linkages and the transient noncovalent
Fig. 9 (a) Photographs of the HPOX 3 self-assemblies with changing
temperature, and (b) their reversible diameter changes at pH 7 between 25
and 85 ꢀC. (Diameter ratio: size/498).
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6 T. J. Measey, R. Schweitzer-Stenner, V. Sa and K. Kornev,
Macromolecules, 2010, 43, 7800–7806.
7 G. Askarieh, M. Hedhammar, K. Nordling, A. Saenz, C. Casals,
A. Rising, J. Johansson and S. D. Knight, Nature, 2010, 465, 236–
238.
8 J. Mondal, X. Zhu, Q. Cui and A. Yethiraj, J. Phys. Chem. C, 2010,
114, 13551–13556.
9 N. l. Carulla, M. Zhou, E. Giralt, C. V. Robinson and C. M. Dobson,
Acc. Chem. Res., 2010, 43, 1072–1079.
hydrogen bonding interactions endow the HPOX aggregates
with pH- and thermal-responsiveness.
As a result, the tentative mechanism for the formation of the
HPOX nanoparticles and the reversible dual stimuli-respon-
siveness can be attributed to the noncovalent interactions of
hydrogen bonds and oxime bonds.
10 Y. Levy and J. N. Onuchic, Acc. Chem. Res., 2005, 39, 135–142.
11 E. N. Salgado, R. A. Lewis, J. Faraone-Mennella and F. A. Tezcan, J.
Am. Chem. Soc., 2008, 130, 6082–6084.
12 Y. Ruff and J.-M. Lehn, Biopolymers, 2008, 89, 486–496.
13 J.-M. Lehn, Chem. Soc. Rev., 2007, 36, 151–160.
14 S. J. Rowan, S. J. Cantrill, G. R. L. Cousins, J. K. M. Sanders and
J. F. Stoddart, Angew. Chem., Int. Ed., 2002, 41, 898–952.
15 V. A. Polyakov, M. I. Nelen, N. Nazarpack-Kandlousy,
A. D. Ryabov and A. V. Eliseev, J. Phys. Org. Chem., 1999, 12,
357–363.
Conclusions
Nonamphiphilic HPOXs were successfully prepared by the
oxime coupling reaction of synthetic trialdehyde and bis-ami-
nooxy, and the functional terminals of the HPOXs could be
modulated by changing the molar feeding ratios of the A2 and B3
1
monomers. H NMR, quantitative 13C NMR, HSQC, HMBC
and FTIR were exploited for analyzing the highly branched
structures. UV-Vis analysis, in conjugation with DLS, TEM,
AFM, SEM and FL confirmed the formation of the HPOX self-
assemblies in the mixed DMSO–H2O solution, with uniform
topology. Through adjusting the structural parameters of the
nonamphiphilic HPOXs, the morphologies of the nanoparticles
varied from sphere to bowl shape due to the alteration in the
intra- and inter-molecular noncovalent interactions. Further-
more, the pH and thermal dual responsiveness of the HPOX self-
assemblies were investigated. The intra- and inter-molecular
driving forces of the HPOX nanoparticles originated from the
noncovalent interactions among the oxime bonds, aminooxy
terminals and amide bonds, rather than the p–p stacking inter-
actions of phenyl groups in the backbone. Primarily, the supra-
molecular interactions between the dynamic oxime bonds and
the alkyl aminooxy groups play a crucial role in the formation of
the HPOX micelles. Moreover, the nonamphiphilic HPOXs
contain a vast number of amide groups, which afford more sites
for multiple hydrogen bonding interactions as well. HPOXs are
orchestrated to construct the novel nonamphiphilic micelles with
stimuli responsiveness. Furthermore, the HPOX self-assemblies
will render us with a profound understanding of the sophisticated
self-assembling behavior of nonamphiphilic biomolecules. This
ongoing work aims to further elucidate the detailed mechanism
of the novel polymeric self-assemblies.
16 J. Liu, W. Huang, Y. Pang, P. Huang, X. Zhu, Y. Zhou and D. Yan,
Angew. Chem., Int. Ed., 2011, 50, 9162–9166.
17 F.-M. Xu, J.-P. Xu, L.-P. Lv and J. Ji, Soft Matter, 2011, 7, 1114–
1120.
18 I. C. Riegel, A. Eisenberg, C. L. Petzhold and D. Samios, Langmuir,
2002, 18, 3358–3363.
19 X. Liu, J.-S. Kim, J. Wu and A. Eisenberg, Macromolecules, 2005, 38,
6749–6751.
20 A. Ebner, L. Wildling, A. S. M. Kamruzzahan, C. Rankl, J. Wruss,
€
C. D. Hahn, M. Holzl, R. Zhu, F. Kienberger, D. Blaas,
P. Hinterdorfer and H. J. Gruber, Bioconjugate Chem., 2007, 18,
1176–1184.
21 R. C. Werlen, M. Lankinen, R. E. Offord, P. August Schubiger,
A. Smith and K. Rose, Cancer Res., 1996, 56, 809–815.
22 T. Zhou, Z. D. Liu, H. Neubert, X. L. Kong, Y. M. Ma and
R. C. Hider, Bioorg. Med. Chem. Lett., 2005, 15, 5007–5011.
23 I. Merino, J. D. Thompson, C. B. Millard, J. J. Schmidt and
Y.-P. Pang, Bioorg. Med. Chem., 2006, 14, 3583–3591.
24 S. Kim, E. Kim, D.-S. Shin, H. Kang and K.-B. Oh, Bioorg. Med.
Chem. Lett., 2002, 12, 895–898.
25 J. M. Hooker, A. Datta, M. Botta, K. N. Raymond and
M. B. Francis, Nano Lett., 2007, 7, 2207–2210.
26 L. Zhang and J. P. Tam, Anal. Biochem., 1996, 233, 87–93.
27 P. E. Dawson and S. B. H. Kent, Annu. Rev. Biochem., 2000, 69, 923–
960.
28 J. A. Borgia and G. B. Fields, Trends Biotechnol., 2000, 18, 243–251.
29 Y. Sohma and S. B. H. Kent, J. Am. Chem. Soc., 2009, 131, 16313–
16318.
30 Y. Jin, L. Song, Y. Su, L. Zhu, Y. Pang, F. Qiu, G. Tong, D. Yan,
B. Zhu and X. Zhu, Biomacromolecules, 2011, 12, 3460–3468.
31 J. Chen, W. Zeng, R. Offord and K. Rose, Bioconjugate Chem., 2003,
14, 614–618.
32 C. J. Hawker, R. Lee and J. M. J. Frechet, J. Am. Chem. Soc., 1991,
113, 4583–4588.
Acknowledgements
33 M. Herrera, G. Manuschek and A. Kettrup, J. Therm. Anal. Calorim.,
2000, 59, 385–394.
34 L. Song, C. Tu, Y. Shi, F. Qiu, L. He, Y. Jiang, Q. Zhu, B. Zhu,
D. Yan and X. Zhu, Macromol. Rapid Commun., 2010, 31, 443–448.
35 M. Herrera, G. Matuschek and A. Kettrup, Chemosphere, 2001, 42,
601–607.
This work is sponsored by the National Natural Science Foun-
dation of China (20974062, 30700175) and the National Basic
Research Program 2009CB930400, the Shuguang Program
(08SG14), the Shanghai Leading Academic Discipline Project
(Project number: B202), and the China National Funds for
Distinguished Young Scientists (21025417).
36 J. Wang, M. Kuang, H. Duan, D. Chen and M. Jiang, Eur. Phys. J. E:
Soft Matter Biol. Phys., 2004, 15, 211–215.
37 W. Cai, G.-T. Wang, Y.-X. Xu, X.-K. Jiang and Z.-T. Li, J. Am.
Chem. Soc., 2008, 130, 6936–6937.
Notes and references
38 Y. Zhou and D. Yan, Angew. Chem., Int. Ed., 2004, 43, 4896–4899.
39 M. Yang, W. Wang, F. Yuan, X. Zhang, J. Li, F. Liang, B. He,
B. Minch and G. Wegner, J. Am. Chem. Soc., 2005, 127, 15107–15111.
40 J.-X. Yang, X.-T. Tao, C. X. Yuan, Y. X. Yan, L. Wang, Z. Liu,
Y. Ren and M. H. Jiang, J. Am. Chem. Soc., 2005, 127, 3278–
3279.
1 L. Wu, J. Lal, K. A. Simon, E. A. Burton and Y.-Y. Luk, J. Am.
Chem. Soc., 2009, 131, 7430–7443.
2 E. Yoshida and S. Kunugi, Macromolecules, 2002, 35, 6665–6669.
3 A. Fegan, B. White, J. C. T. Carlson and C. R. Wagner, Chem. Rev.,
2010, 110, 3315–3336.
4 E. N. Salgado, R. J. Radford and F. A. Tezcan, Acc. Chem. Res.,
2010, 43, 661–672.
5 B. Kokona, A. M. Kim, R. C. Roden, J. P. Daniels, B. J. Pepe-
Mooney, B. C. Kovaric, J. C. de Paula, K. A. Johnson and
R. Fairman, Biomacromolecules, 2009, 10, 1454–1459.
41 C. B. Minkenberg, L. Florusse, R. Eelkema, G. J. M. Koper and
J. H. van Esch, J. Am. Chem. Soc., 2009, 131, 11274–11275.
42 P. T. Corbett, J. Leclaire, L. Vial, K. R. West, J.-L. Wietor,
J. K. M. Sanders and S. Otto, Chem. Rev., 2006, 106, 3652–3711.
43 J.-M. Lehn, Science, 2002, 295, 2400–2403.
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