Betaine ester-shell functionalized hyperbranched polymers for potential antimicrobial usage: Guest loading capability, pH controlled release and adjustable compatibility

Article abstract of DOI:10.1016/j.polymer.2014.10.020

Betaine ester-shell functionalized hyperbranched polyethylenimines (BEHPEI) were synthesized by Menschutkin reaction between per-N-methylated polyethylenimine (MeHPEI) and alkyl bromoacetate. BEHPEI could play dual antimicrobial roles that were, the betaine ester-shell acted as contact-based antibacterial polycations and the drug-loaded BEHPEI could controllably release the drugs due to the cleavage of the betaine ester groups under weak alkaline condition. The BEHPEI exhibited high transport capacities that per gram of BEHPEI could encapsulate 0.24-2.67 g of dyes or drugs. The model drug release experiment employing methyl orange (MO) showed that the drug release of MO-loaded BEHPEI complex occurred slowly in weak alkaline solutions and the release rate was controlled by varying pH, while the complex kept very stable under weak acid condition (pH = 3.0-7.0). BEHPEI generated from long chain alkyl bromoacetate was compatible with organic resins implying the possible usage in antimicrobial fibers and coatings. Another BEHPEI obtained from short-chain alkyl bromoacetate was water soluble and maybe used in lotion formula. The hydrolysis of BEHPEI afforded zwitterionic shell.

Full text of DOI:10.1016/j.polymer.2014.10.020

Polymer 55 (2014) 6261e6270
Contents lists available at ScienceDirect
Polymer
journal homepage: www.elsevier.com/locate/polymer
Betaine ester-shell functionalized hyperbranched polymers
for potential antimicrobial usage: Guest loading capability,
pH controlled release and adjustable compatibility
Xin Zhou, Yongyue Chen, Jin Han*, Xuedong Wu*, Gang Wang, Daoyi Jiang
Key Laboratory of Marine Materials and Related Technologies, Zhejiang Key Laboratory of Marine Materials and Protective Technologies,
Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences, Ningbo 315201, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Betaine ester-shell functionalized hyperbranched polyethylenimines (BEHPEI) were synthesized by
Menschutkin reaction between per-N-methylated polyethylenimine (MeHPEI) and alkyl bromoacetate.
BEHPEI could play dual antimicrobial roles that were, the betaine ester-shell acted as contact-based
antibacterial polycations and the drug-loaded BEHPEI could controllably release the drugs due to the
cleavage of the betaine ester groups under weak alkaline condition. The BEHPEI exhibited high transport
capacities that per gram of BEHPEI could encapsulate 0.24e2.67 g of dyes or drugs. The model drug
release experiment employing methyl orange (MO) showed that the drug release of MO-loaded BEHPEI
complex occurred slowly in weak alkaline solutions and the release rate was controlled by varying pH,
while the complex kept very stable under weak acid condition (pH ¼ 3.0e7.0). BEHPEI generated from
long chain alkyl bromoacetate was compatible with organic resins implying the possible usage in anti-
microbial fibers and coatings. Another BEHPEI obtained from short-chain alkyl bromoacetate was water
soluble and maybe used in lotion formula. The hydrolysis of BEHPEI afforded zwitterionic shell.
© 2014 Elsevier Ltd. All rights reserved.
Received 15 May 2014
Received in revised form
15 September 2014
Accepted 7 October 2014
Available online 14 October 2014
Keywords:
Betaine ester-shell
Supramolecular encapsulation
pH controlled release
1. Introduction
material largely depends on property of matrix and compatibility
between matrix and drugs. However, because the majority of
Microbial contamination and the associated risk are great
challenges towards medical implants [1e4], textiles [5,6], food and
water security [7e9] and biosensors [10,11]. Till now, antimicrobial
materials employing various protection mechanisms have been
developed. Among them, contact-based and release-based anti-
microbial materials for killing or inhibiting bacteria have gained
significant attention.
One typical contact-based antimicrobial material is polycations
which can cause damage to the outer membrane of bacteria by
interacting with the anionic lipopolysaccharide. However, many
contact-based polycations suffer from low sensitivity towards
Gram-negative bacteria [12e16].
antimicrobial drugs are water soluble compounds, low compati-
bility between low polar organic polymer matrix and high polar
drugs leads to low drug-loading capacity and uncontrolled release.
To solve this problem, various amphiphilic polymer capsules for
drug-loading have been developed to enhance compatibility
[21e24].
Physical aggregates of amphiphilic macromolecules with lipo-
some or micelle structures are usually used as drug-delivery sys-
tems in the past decades [25]. However, these structures are
unstable under environment effects like shear force, temperature
and pressure [26]. Then, dendritic polymers possessing stable
coreeshell structures emerged as novel materials for supramolec-
ular encapsulation [27e30]. Among them, pH-responsive, espe-
cially acid-responsive, dendritic polymers are promising due to the
potential application in in-vivo cancer therapy (faintly acid envi-
ronment) [31e34]. Haag et al. [35] reported that HPEI linked with
poly (ethylene glycol) shells by imine bonds showed high loading
capacities for polar dyes and could rapidly release dye molecules
under acidic condition (pH ¼ 5). However, there are occasions
where acid stability or alkaline lability is needed. For example,
Release-based antimicrobial materials have long-term antimi-
crobial effects because of slow release of drugs such as antibiotics,
algaecides, preservatives and silver nanoparticles encapsulated in
polymer matrix [17e20]. Controlled release property of this sort of
* Corresponding authors. Fax: þ86 574 86685159.
E-mail addresses: hj@nimte.ac.cn (J. Han), xdwu@nimte.ac.cn (X. Wu).
http://dx.doi.org/10.1016/j.polymer.2014.10.020
0032-3861/© 2014 Elsevier Ltd. All rights reserved.

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X. Zhou et al. / Polymer 55 (2014) 6261e6270
weak alkaline environment like seawater and human blood and
intestines could call for suitable basic degradability of capsules to
release drugs, while weak acidic fresh water demands acid stability.
Antimicrobial materials combining contact-based and released-
based mechanisms have been reported in several works
[13,36e38]. For example, Sen et al. embedded silver bromide into
amphiphilic pyridinium polymer matrix to achieve dual actions
[36]. Cohen et al. used layer-by-layer technique to fabricate a
coating embedded with silver and cationic nanoparticles [13].
These works focused on fabricating antibacterial coatings, while
dual-function drug capsules have never been reported.
In this work, weak base-labile betaine ester-shell was utilized to
play a contact killing role [39] and to form a labile coreeshell
structure for drug loading and release [40]. Both dyes and drugs
were employed to study the pH-responsive property by means of
UVeVis spectrometry. Long chain shell could endow dye-loaded
BEHPEI good compatibility with organic polymer matrix for likely
coating application, while short chain shell make the polymer
water soluble for possible antibacterial lotion usage.
magnesium sulfate, ethanol was removed under reduced pressure
and MeHPEI (9.68 g, 73.1%) was collected as a yellow viscous
material.
2.3.2. Synthesis of dodecanyl bromoacetate
Freshly distilled bromoacetyl bromide (26 g, 130 mmol) and
dried CH2Cl2 (80 mL) were mixed in a 250 mL round-bottom flask in
an iceewater bath. A CH2Cl2 (50 mL) solution containing dodecanol
(18.63 g, 100 mmol) and N,N-diisopropylethylamine (16.77 g,
130 mmol) was dropwise added to the flask with vigorous stirring
for 2 h. The reaction was carried out in an iceewater bath for 4 h
and then at ambient temperature for 20 h. The precipitates were
filtered off, and the filtrate was washed in sequence with 1 M HCl,
1 M NaOH, and saturated NaCl aqueous solution. The organic phase
was separated and dried over magnesium sulfate. After removal of
CH2Cl2 via rotary evaporation, the residual was dried in vacuo at
room temperature overnight to afford dodecanyl bromoacetate
(26.96 g, 70.0%).
2.3.3. Synthesis of betaine ester wrapped hyperbranched
polyethylenimine (BEHPEI)
2. Experimental part
MeHPEI (2.57 g) and DMF (5 mL) were mixed in a 100 mL round-
bottom flask. Dodecanyl bromoacetate (26.51 g) was dissolved in
DMF (25 mL) and dropwise added into the flask. Then, the flask was
stirred at room temperature for 48 h. Then, DMF was removed
under reduced pressure. The residual was cooled to 0 ꢀC, and excess
dodecanyl bromoacetate was retrieved carefully by a syringe. The
residual was dissolved with 3 mL CHCl3, and poured into 20 mL cold
ethyl ether (0 ꢀC) to precipitate a brown solid. After dried in vac-
uum, BEHPEI was obtained (8.7 g, 73.7%).
2.1. Materials
Hyperbranched polyethylenimine (Mw
¼
25,000 Da (LS),
Mn ¼ 10,000 Da (GPC)) was purchased from SigmaeAldrich.
Ethanol, formic acid, formaldehyde, bromoacetyl bromide, dodec-
anol, allyl alcohol, methyl orange, methyl blue (MB), fluorescein
sodium (FS), eosin Y (EY), congo red (CR), methyl violet (MV), cal-
cium hydride and anhydrous magnesium sulfate were obtained
from Aladdin Chemical Co. China. Sodium sorbate (SS) and tetra-
cycline (TC) were purchased from Chengdu Ai Keda Chemical
Technology Co. Diethyl ether, chloroform, methylene dichloride,
N,N-dimethylformamide, N,N-diisopropylethylamine, and sodium
hydroxide were from Sinopharm Chemical Reagent Co. Dichloro-
methane and N,N-diisopropylethylamine were dried over CaH2
before use. All other reagents were used as received.
2.3.4. Synthesis of water-soluble allyl BEHPEI
Allyl bromoacetate was obtained from allyl alcohol and bro-
moacetyl bromide. Then, allyl bromoacetate (4.2 g) and MeHPEI
(1.0 g) were used to prepare the water-soluble allyl BEHPEI. The
reaction was carried out in 10 mL DMF. After 48 h, the reaction
solution was poured into 100 mL diethyl ether. The precipitates
were collected and dried in vacuum to give a yellow solid (2.99 g,
96.7%).
2.2. Instruments
1H and 13C NMR measurements were carried out on an Avance
III 400 MHz spectrometer at room temperature. The samples were
dissolved in CDCl3 or D2O. Tetramethylsilane was used as an in-
ternal reference. Quantitative 13C spectra were recorded using in-
verse gated decoupling pulse sequence to avoid any influence of the
Nuclear Overhauser effect. UVeVis spectroscopic analysis was
recorded on a Lambda 950 UVeVis spectrophotometer. FTIR spec-
trum analysis was carried out on a Nicolet 7600 spectrometer.
2.3.5. Determination of the encapsulating capabilities for BEHPEI
Several dyes were dissolved in deionized water to prepare so-
lutions with concentrations of 1 ꢁ 10ꢂ6 mol Lꢂ1, 2 ꢁ 10ꢂ6 mol Lꢂ1
,
5 ꢁ 10ꢂ6 mol Lꢂ1, 10 ꢁ 10ꢂ6 mol Lꢂ1, 20 ꢁ 10ꢂ6 mol Lꢂ1 and
30 ꢁ 10ꢂ6 mol Lꢂ1. By means of UVeVis spectra, the absorption
maximums (A) of dyes' solutions were detected and plotted versus
the concentrations. Linear relationships between A and concen-
tration were observed, resulting in a series of standard work curves.
In the dye transportation experiment, the aqueous solutions of
CR and MO were 2 ꢁ 10ꢂ3 mol Lꢂ1, the aqueous solutions of FS, EY,
MB and MV were 1 ꢁ 10ꢂ3 mol Lꢂ1, and the CHCl3 solutions of
BEHPEI was 0.40 g Lꢂ1. Typically, equal volumes of a chloroform
solution of BEHPEI and a dye solution were pipetted into a vial and
the vial was vibrated fiercely. After phase separation, it was
observed that the nether chloroform phase was dyed. The upper
water phase was analyzed by UVeVis spectroscopy to calculate the
amount of the remaining dyes in the aqueous phase according to
the corresponding standard curve, and thus the amount of the dye
transported by BEHPEI was known.
2.3. Methods
2.3.1. Synthesis of hyperbranched per-N-methylated
polyethylenimine (MeHPEI)
Hyperbranched polyethylenimine (10 g, 0.1 mol) was dissolved
in distilled water (75 mL) and charged into a round bottom two-
neck flask (500 mL). The flask was equipped with a reflux
condenser on top of which an oil bubbler was fitted. Excess formic
acid (245 g, 5.3 mol) and formalin (36 g, 1.2 mol) were successively
added into the flask. After being purged with N2 gas, the reaction
mixture was kept at 100 ꢀC for 5 days under stirring. All the vola-
tiles were removed under reduced pressure. Then, 2 N NaOH
aqueous solution was added and the formed sodium formate was
precipitated by the addition of ethanol and removed by filtration.
The solvent was removed via rotary evaporation and the residue
was added with 200 mL ethanol again. After dried over anhydrous
2.3.6. Coloring resins by the dye-loaded BEHPEI via solution
blending
Two chloroform solutions of dye-loaded BEHPEI were added
with PMMA, respectively. When the solutions became homoge-
nous, they were poured onto petri dishes, dried in a fuming

X. Zhou et al. / Polymer 55 (2014) 6261e6270
6263
cupboard for one week and finally dried in vacuum for 24 h to give
transparent and homogenous films. In the control experiments,
dyes were directly added into the chloroform solutions of PMMA to
produce films by following the same procedure.
because it has been proved that they own strong antibacterial ac-
tivity and interesting degradation behavior [39,40]. Compared with
normal ester bonds, due to the electron-withdrawing effect of
ammonium group adjacent to ester bond, betaine ester bonds un-
dergo much faster cleavage in alkali mediums, but much slower
cleavage under acid conditions [40]. Herein, these groups were
formed at the outside of HPEI, facilitating them to contact bacteria.
As outlined in Scheme 1, EschweilereClark reaction of HPEI yielded
MeHPEI, and then highly efficient Menschutkin reaction between
MeHPEI and dodecanyl bromoacetate was performed to generate
BEHPEI. Polar aprotic solvent, DMF, was chosen to make the
nucleophilic reaction proceed rapidly at room temperature and
achieve nearly quantitative conversion of methylamino groups into
betaine ester groups.
2.3.7. pH-controlled release of dyes
The nether CHCl3 solution of MO-loaded BEHPEI obtained in the
dye transportation experiment was dried on a rotary evaporator
and then in vacuum for 24 h to yield the supramolecular complex as
brown solid, which were subsequently ground into powders. The
powder composed of MO and BEHPEI was immersed in a series of
buffer solutions of various pH values from 1.0 to 10.4. The release
amount of MO at different times was monitored by UVeVis
spectroscopy.
In the 1H NMR spectrum of MeHPEI (Fig. 1, a1), the peak at
2.2 ppm was attributed to the proton signal of the formed methyl
groups resulted from the EschweilereClark reaction. As a conse-
quence of Menschutkin reaction, in the FTIR spectrum of BEHPEI,
the band at 1740 cmꢂ1 resulted from the stretching vibration of
eC]O groups (Fig. 2), and in the 1H and 13C NMR spectra of
BEHPEI (Fig. 1, c1 and c2), the proton signals ascribed to the
3. Results and discussion
3.1. Synthesis of BEHPEI
MeHPEI was chosen on accounts of its high polarity in favor of
high encapsulating capacity. Betaine ester groups play a key role,
Scheme 1. Synthesis of coreeshell structured base-labile BEHPEI.

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X. Zhou et al. / Polymer 55 (2014) 6261e6270
dodecanyl groups appeared at 1.3 ppm, the proton signals
assigned to the original PEI moiety emerged at 3.0e5.0 ppm, and
all the carbon signals of dimethylamino groups at 44.0 ppm shif-
ted to 51.5 ppm due to the formation of dimethylammonium
groups. These results confirmed that BEHPEI was successfully
prepared and the conversion of dimethylamino and methylamino
groups into betaine ester groups was nearly quantitative. A
detailed examination on the BEHPEI structure was presented in
the Supporting Information (Fig. 2).
3.2. Encapsulation capacities of BEHPEI for several dyes and drugs
The structures of the employed dyes and drugs were displayed
in Fig. 3. According to LamberteBeer law, when wavelength of
Fig. 1. 1H NMR spectra of MeHPEI (a1), dodecanyl bromoacetate (b1) and BEHPEI (c1); 13C NMR spectra of MeHPEI (a2), dodecanyl bromoacetate (b2) and BEHPEI (c2).

X. Zhou et al. / Polymer 55 (2014) 6261e6270
6265
Fig. 4. Standard curves of dyes and drugs.
Fig. 2. FTIR spectra of MeHPEI (a), dodecanyl bromoacetate (b) and BEHPEI (c).
concentrations were examined by means of UVeVis spectroscopy
and the results were presented in Fig. 4 and Table 1.
Typically, a chloroform solution of BEHPEI was fiercely mixed
with a guest's aqueous solutions and after phase separation, the
chloroform solution was found to be colorized (Fig. 5). It was
explained that BEHPEI accommodated the polar guest molecules
within its polar core through electrostatic interaction or hydro-
philic interaction [41]. The remaining amount of the guest in the
aqueous phase was analyzed using UVeVis spectroscopy. The
incident light, liquid layer thickness and temperature of solution
were constant, maximum absorbance of solution (A) in a certain
range was proportional to concentration following an equation:
A ¼ εcl. Here, c denoted the concentration; l was the thickness of
liquid layer which was 1 cm, the size of cuvette; ε was the molar
absorption coefficient. In order to determine the proportions be-
tween A and c, dyes' or drugs' aqueous solutions of different
Fig. 3. Structures of the employed dyes and drugs.

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X. Zhou et al. / Polymer 55 (2014) 6261e6270
Table 1
Table 2
Standard curve equations and molar absorption coefficients of dyes and drugs.
Encapsulation capacities of BEHPEI for different dyes and drugs.
Dye and drug ε ꢁ 10ꢂ6 [L molꢂ1 cmꢂ1
]
R2a
Standard curve equation
Dye and drug
Encapsulation ꢁ 10ꢂ3 [mol gꢂ1
]
Encapsulation [g gꢂ1
]
FS
0.0198
0.0120
0.0804
0.0148
0.0290
0.0181
0.0076
0.0130
0.9976 A ¼ 0.0198c
0.9991 A ¼ 0.0120c
0.9998 A ¼ 0.0804c
0.9992 A ¼ 0.0148c
0.9999 A ¼ 0.0290c
0.9997 A ¼ 0.0181c
0.9994 A ¼ 0.0076c
0.9996 A ¼ 0.0130c
FS
1.09
2.29
1.79
3.83
1.94
2.78
1.25
1.82
0.41
1.83
1.24
2.67
0.79
0.91
0.56
0.24
MB
EY
CR
MV
MO
SS
MB
EY
CR
MV
MO
SS
TC
TC
a
R is the linear correlation coefficient.
As shown in Fig. 7b, the explanation was described as follows.
The supramolecular encapsulation included two kinds of mode,
intramolecular and intermolecular encapsulation [42]. The
remaining tertiary amino groups were quickly transformed into
quaternary ammonium groups under acid condition. The intensi-
fied electrostatic repulsion effect caused the disassembly of some
aggregates of the supramolecular complexes. As a consequence,
some MO molecules between the BEHPEI macromolecules were
initially released. The 1H spectrum of the BEHPEI after immersion in
weak acid solution (pH ¼ 3.4 and 5.0) did not vary much (Fig. 8b),
indicating that the betaine ester groups were resistant to acid hy-
drolysis, and thus most of the MO molecules hold internally and
firmly by the BEHPEI could not enter the solution in a very long
time. Moreover, by lowering the pH value to 1.0, pH indicative red
was seen and hydrolysis rate was remarkably expedited. None-
theless, the release amount under strong acid condition in 21 d
merely equaled that at pH ¼ 8.2 approximately. Therefore, the
BEHPEI possessed weak acid stability (pH ¼ 3.0e7.0) and alkaline
hydrolysis property, which may be utilized in different pH occa-
sions, such as weak acid freshwater, weak alkaline ocean and so on.
results of the transportation capacities were listed in Table 2.
BEHPEI exhibited excellent encapsulation capacities towards these
guests. For CR and MB, 1 g of BEHPEI could encapsulate 2.67 and
1.83 g, respectively, more than the amounts for other guests. It was
explained that there were two or more negatively charged eSO3ꢂ
groups in CR and MB, so they had stronger electrostatic interaction
with BEHPEI. Two drugs, antibiotic tetracycline hydrochloride and
antiseptic sodium sorbate, were also effectively encapsulated,
implying the successful acquisition of two supramolecular com-
plexes possessing two antibacterial weapons, the betaine ester
groups and the loaded drugs (Table 2).
3.3. Controlled release of encapsulated dyes by pH stimuli
Photographs of MO-encapsulated BEHPEI immersed in different
pH solutions are presented in Fig. 6. Under neutral condition
(pH ¼ 7.0), the hydrolysis proceeded very slowly, and an MO con-
centration in the buffer solution less than 10 m
mol Lꢂ1 was detec-
ted. In weak alkaline medium, pH ¼ 8.2, which was in the pH range
of natural seawater, the hydrolysis became faster, the buffer solu-
tion gradually turned brown and the MO concentration reached
3.4. Compatibility of the supramolecular complex with resins
71
m
mol Lꢂ1 in 21 d. The general mechanism was that the alkaline
Compatibility of the supramolecular complex with polymer
resins should be taken into account to fabricate antibacterial ma-
terials, because it was associated with drug-carrying amount and
controllability of release. According to the literatures, a number of
antibacterial materials employed polymer resins containing ester
bonds, such as poly(lactic-co-glycolic acid), poly(ε-caprolactone),
PMMA and so on [43,44]. Here, we chose PMMA as model resins to
test the compatibility of the supramolecular complex, because
PMMA is highly transparent, could make the contrast more distinct.
Solution blending method for film formation was adopted.
PMMA resin was dissolved in the chloroform solution of MO-loaded
BEHPEI and dried in a fume hood to give a uniform and transparent
yellow film (Fig. 9 a2), while another film without using BEHPEI
prepared in the same way was hardly dyed (Fig. 9 a1). This
demonstrated that the supramolecular complex was compatible
with PMMA and the BEHPEI greatly enhanced the dispersion of MO.
Besides, the film containing the complexes showed resistance to-
wards water immersion washing (Fig. 9 b1). The other film without
hydrolysis of betaine ester bonds led to the departure of hydro-
phobic alkyl shell which was proved by the disappearance of the
signals at 1.25 ppm ascribed to the long chain alkyl protons (Fig. 8a),
gave a hydrophilic zwitterionic shell, made the powders gradually
dissolved into the buffer solutions, and in the meantime, MO
molecules diffused into water (Fig. 7a). This slow release behavior
may be utilized in the design of marine antifouling coating. The
hydrolysis speed was accelerated as pH increased to 9.2 and 10.4,
and consequently, remarkably visible color change occurred early
in 1 d and 4 h, respectively. In 21 d, at pH ¼ 10.4, the MO concen-
tration achieved 250
m
mol Lꢂ1 and the amount of the powders
obviously reduced. Hydrolysis behaviors in relatively weak acid
medium (pH ¼ 3.4 and 5.0) were interesting. The buffer solutions
turned slightly yellow in 1 h, earlier than the time when remark-
able color change in the alkaline hydrolysis case was observed,
however, the color depth did not intensify as could be seen and the
final MO concentrations in 21 d were less than 25 m .
mol Lꢂ1
Fig. 5. Photographs of phase transferddcomparison of chloroform and BEHPEI chloroform solution when mixed with FS (A), MB (B), EY (C), CR (D), MV (E) and MO (F) aqueous
solution respectively (upper layer: water phase, bottom layer: chloroform phase, left: without BEHPEI, right: with BEHPEI).

X. Zhou et al. / Polymer 55 (2014) 6261e6270
6267
Fig. 6. Photographs of MO-encapsulated BEHPEI immersed in different pH solutions. (Left pictures: from top to bottom, soaking time was 10 min, 30 min, 60 min, 4 h, 8 h, 1 d, 3 d,
10 d and 21 d; from left to right, pH value of the solution was 1.0, 3.4, 5.0, 7.0, 8.2, 9.2 and 10.4. Right pictures: dyes concentration of soaking time from 10 min to 21 d (a) and 10 min
to 1 d (b)).
Fig. 7. Schematic showing of the evolution of BEHPEI immersed in alkaline solution (a) and weak acidic solution (b).

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X. Zhou et al. / Polymer 55 (2014) 6261e6270
using BEHPEI had many visible dye particles lying on the film
surface and thus the dye could enter water easily (Fig. 9 b2). After
immersing the film in water for 1 h, water exhibited yellow color,
and in 2 days, the concentration of MO dye in water reached
0.039 mmol/L. Examination of the washing water by means of
UVeVis spectroscopy confirmed that trace amount of dyes was
released by the BEHPEI-containing sample (Fig. 9c). Thus, the su-
pramolecular complex enhanced the dispersion of guests.
3.5. Water-soluble allyl BEHPEI
Long chain alkyl shell offered BEHPEI the compatibility with
resins, while short chain shell endowed BEHPEI with water solu-
bility. Because allyl group normally show its proton signals at
5.6e6.0 ppm and it could make the study of hydrolysis process
easier, allyl 2-bromoacetate was used here. The Menschutkin re-
action between allyl 2-bromoacetate and MeHPEI yielded the
target polymer (Scheme 2). Solubility test showed that it could be
mixed with water in all proportions at 29 ꢀC. The dissolution speed
was fast. 5 mL water was added into a 25 mL beaker, and was stirred
by a magnetic stirrer at a speed of 200 r/min. A piece of 0.2 g
polymer was put into the beaker and it was totally dissolved in
105 s. Immersing the polymer in a buffer solution of pH ¼ 8.4 led to
Fig. 8. (a) 1H NMR spectrum of hydrolysis product of BEHPEI after long time immer-
sion in alkaline solution (D2O); (b) 1H NMR spectrum of BEHPEI after immersion in
weak acid solution (pH ¼ 3.4 and 5.0) in 21 d (CDCl3); (c) 1H NMR spectrum of pristine
BEHPEI (CDCl3).
Fig. 9. Photographs of MO-coloring PMMA membranes placed on a paper (a1 without BEHPEI, a2 with BEHPEI) and immersed in water for 2 d (b1 without BEHPEI, b2 with BEHPEI);
the corresponding UVeVis spectra of aqueous solution after immersing dyed PMMA films (c).

X. Zhou et al. / Polymer 55 (2014) 6261e6270
6269
Scheme 2. Synthetic route of water-soluble allyl BEHPEI.
the carboxybetaine-functionalized product in 24 h (Fig. 10). The
short chain poly (betaine esters) has been found excellent activity
against bacteria like Escherichia coli K12 [45], and the carbox-
ybetaine groups are nontoxic to cells and resistant to nonspecific
protein adsorption [46], so the prepared water soluble allyl BEHPEI
maybe found usage in hand washing lotion, mouth rinse and etc.
4. Conclusion
A general avenue to prepare betaine ester-shell functionalized
hyperbranched polyethylenimine (BEHPEI) in large scale was pre-
sented. The BEHPEI possesses these functions: firstly, betaine ester-
shell could act as antibacterial polycations; secondly, the coreeshell
structure of opposite polarity could encapsulate large amount of
antibiotic TC and antiseptic SS (0.24 and 0.56 g gꢂ1, respectively);
thirdly, betaine ester shell could be controllably degraded under
weak alkaline conditions (pH ¼ 7e9) to realize a controlled release
operation while kept stable under neutral and weak acid environ-
ments (pH ¼ 3.0e7.0); fourthly, varying the chain length of betaine
ester-shell could adjust the compatibility with organic resins or
water for different usages. Based on these investigations, antimi-
crobial applications of drug-loaded BEHPEI in biodegradable poly-
mer coatings and lotions are in process in our lab.
Fig. 10. 1H NMR spectra of allyl 2-bromoacetate (a), water-soluble allyl BEHPEI (b), allyl
BEHPEI immersed in alkaline solution for 10 h (c), BEHPEI immersed in alkaline so-
lution for 24 h (d) and allyl alcohol (e) in D2O.

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The authors acknowledge the financial support from Ningbo
Nature Science Foundation (2013A610018), Ningbo Special Project
(2013B6012), National Nature Science Foundation (51303192), State
Key Program of National Natural Science of China (51335010), Na-
tional Basic Research Program of China (2014CB643302), and
Ministry of Science and Technology of China (1106).
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Appendix A. Supplementary data
Supplementary data related to this article can be found online at
http://dx.doi.org/10.1016/j.polymer.2014.10.020.
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