Ionic liquid-doped and p-NIPAAm-based copolymer (p-NIBIm): Extraordinary drug-entrapping and -releasing behaviors at 38-42 °c

Article abstract of DOI:10.1039/c4ra03736g

Ionic liquid (IL)-doping of the temperature responsive p-NIPAAm was achieved by radical copolymerization of N-isopropyl acryl amide (NIPAAm; 90 mol%) and 1-butyl-3-vinylimidazolium bromide ([BVIm]Br; 10 mol%) to give a new temperature responsive copolymer (p-NIBIm). The as-prepared p-NIBIm copolymer showed a highly increased zeta potential value and optimal LCST (lower critical solution temperatures) value, respectively, +9.8 mV at pH = 7 and 38.2 °C, compared to those (+0.3 mV at pH = 7 and 32.1 °C) of p-NIPAAm. The temperature-dependent size change of the p-NIBIm micelles was determined in the range from 25 to 45 °C by SEM under dry conditions and by a zeta sizer under wet conditions, showing a certain size contraction from 253 ± 12.1 to 90.5 ± 7.8 nm in diameter (about 95.4% of volume contraction). The thermo-sensitive behavior to entrap BSA protein at body temperature (37 °C) and to release the protein between 38-42 °C (near the LCST) were also tested by sizing of the complexes of p-NIBIm/BSA using a zeta sizer and also by a colorimetric assay (Bio-Rad DC Protein Assay), resulting in a maximum entrapment of 1.02 mg BSA for 1.0 mg of the polymer at body temperature (37 °C) and in a maximum release of 0.73 mg BSA for 1.0 mg of the polymer (about 73% release of the entrapped amount) in the temperature range of 38-42 °C. Toxicity of the p-NIBIm micelles (in the range of <0.125 mg mL^-1) without drug for human embryonic kidney (HEK 293) cells was minimal in vitro. These results revealed that the IL-doped and temperature responsive co-polymeric systems have a very high applicability as a novel delivery system for charged (or polar) molecules as a natural (or synthetic) drug and DNA. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ra03736g

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Ionic liquid-doped and p-NIPAAm-based
copolymer (p-NIBIm): extraordinary drug-
entrapping and -releasing behaviors at 38–42 C†
Cite this: RSC Adv., 2014, 4, 26738
ꢀ
ab
Jae-Won Seo,ab Ji-Young Hwangab and Ueon Sang Shin
*
Ionic liquid (IL)-doping of the temperature responsive p-NIPAAm was achieved by radical copolymerization of
N-isopropyl acryl amide (NIPAAm; 90 mol%) and 1-butyl-3-vinylimidazolium bromide ([BVIm]Br; 10 mol%) to
give a new temperature responsive copolymer (p-NIBIm). The as-prepared p-NIBIm copolymer showed a
highly increased zeta potential value and optimal LCST (lower critical solution temperatures) value,
respectively, +9.8 mV at pH ¼ 7 and 38.2 ꢀC, compared to those (+0.3 mV at pH ¼ 7 and 32.1 ꢀC) of p-
NIPAAm. The temperature-dependent size change of the p-NIBIm micelles was determined in the range
from 25 to 45 ꢀC by SEM under dry conditions and by a zeta sizer under wet conditions, showing a certain
size contraction from 253 ꢁ 12.1 to 90.5 ꢁ 7.8 nm in diameter (about 95.4% of volume contraction). The
thermo-sensitive behavior to entrap BSA protein at body temperature (37 ꢀC) and to release the protein
between 38–42 ꢀC (near the LCST) were also tested by sizing of the complexes of p-NIBIm/BSA using a
zeta sizer and also by a colorimetric assay (Bio-Rad DC Protein Assay), resulting in a maximum entrapment
of 1.02 mg BSA for 1.0 mg of the polymer at body temperature (37 ꢀC) and in a maximum release of 0.73
mg BSA for 1.0 mg of the polymer (about 73% release of the entrapped amount) in the temperature range
of 38–42 ꢀC. Toxicity of the p-NIBIm micelles (in the range of <0.125 mg mLꢂ1) without drug for human
embryonic kidney (HEK 293) cells was minimal in vitro. These results revealed that the IL-doped and
temperature responsive co-polymeric systems have a very high applicability as a novel delivery system for
charged (or polar) molecules as a natural (or synthetic) drug and DNA.
Received 24th April 2014
Accepted 5th June 2014
DOI: 10.1039/c4ra03736g
www.rsc.org/advances
applicability as a delivery system due to the too low LCST (below
body temperature), a tedious drug-releasing prole in a wide
1 Introduction
temperature range below body temperature and too low drug-
carrying ability (about <0.1 mg drug per 1.0 mg polymer).8 To
resolve these problems, the hydrophilic p-NIPAAm segments
have been incorporated with relatively hydrophobic polymer
segments to prepare thermo-responsive p-NIPAAm-based
copolymers. The prepared copolymers exhibited some addi-
tional advantages for various biomedical applications, for
example, a possible tuning of the LCST between 37 (body
temperature) and 42 ꢀC (clinical hyperthermic temperature)18
and creating other property (pH-sensitive),19,20 but without a
satisfactory improvements in the drug loading level (<36 wt%),
the pH-dependant nature of LCST, and the drug-releasing
pattern of co-polymeric drug-carriers.19,21–25
To achieve more efficient drug delivery exactly to a target site,
we hypothesized that co-polymeric drug-carriers (or micelles)
have to form stable complexes with drug-molecules for
improving drug-carrying ability, to have suitable and constant
LCSTs (around 38–40 ꢀC) unaffected by encapsulated drugs and
surrounding pH, and to exhibit faster response (for example,
within several seconds) for exact drug-releasing at a target
temperature range. In this way, the introduction of ionic
moieties into a polymer chain will be an effective strategy,
Environmentally sensitive materials have recently attracted
considerable attention due to their biomedical applications
owing to the reversible responses to external stimuli, such as
temperature,1 pH,2 ionic strength,3 electric eld,4 magnetic
eld,5 light,6 etc.7–10 Among the external stimuli responsive
materials, temperature- or pH-stimuli responsive polymers have
been most widely investigated, because these two factors of
body tissue could be changed by many diseases and easily
regulated by external induction.7–14 Poly(N-isopropylacrylamide)
(p-NIPAAm) is one of the most well-known thermoresponsive
polymers and has been extensively studied for biomedical
applications owing to the reversible thermoresponsive phase
transition from a hydrated random coil (or a swelled globule) to
a deswelled compact globule at the lower critical solution
temperature (LCST ¼ 32).15–17 However, p-NIPAAm has limited
aInstitute of Tissue Regeneration Engineering (ITREN), Dankook University, South
Korea. E-mail: usshin12@dankook.ac.kr; Fax: +82 41 559 7911; Tel: +82 41 550 3691
bDepartment of Nanobiomedical Science & BK21 PlUS NBM Global Research Center for
Regenerative Medicine, Dankook University, Cheonan, 330-714, Republic of Korea
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ra03736g
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because the charged groups within the polymer chain can was heated to 100 ꢀC for 24 hours by stirring. A dark brown
induce increasing the LCST over body temperature up to clinical viscous residue was obtained aer the complete evaporation of
hyperthermic temperature and forming stable complexes with the volatile parts. The organic salt as a monomeric product, 1-
negatively (or positively) charged guest molecules via ionic butyl-3-vinylimidazolium bromide ([BVIm]Br), was extracted
interactions.26–30 However if the charge density within the with dichloromethane from the aqueous solution. 5.92 g of a
temperature-stimulus responsive polymers is easily affected by yellowish viscous liquid was obtained aer washing the organic
the external pH values (especially for the cases of polymers with phase with distilled water (DW), evaporation of the organic
amine functional groups), their LCST values can also undergo a solvent, and complete vacuum drying. The crude product
change, leading to a failure to exactly deliver drug molecules to obtained was puried by ash column chromatography (MC/
the target site.31 Therefore the existence of permanently (or pH- methanol) to give >90 wt% yield.
independently) ionic moieties within a polymer chain that are
not affected by the surrounding pH condition will be essential 2.3 Synthesis of the IL-doped copolymer, poly(NIPAAm-co-
to transfer drug molecules exactly to the target site with a BVIm) or p-NIBIm
certain temperature and to reduce response times of drug
carriers (or micelles) for drug-releasing.
Here, we prepared a partially positive charged and tempera-
ture responsive co-polymer by radical copolymerization of N-
p-NIPAAm-based and partially cationic copolymer with N-butyl
imidazolium moieties, poly(NIPAAm-co-BVIm), was synthesized,
as shown in Scheme 1. 1.13 g (10 mmol) of NIPAAm and 0.30 g (1
mmol) of 1-butyl-3-vinylimidazolium bromide ([BVIm]Br) were
isopropyl acryl amide (NIPAAm) and 1-butyl-3-vinylimidazolium
dissolved in 40 mL of DW, and then 10 mL (0.5 mmol) of
bromide ([BVIm]Br). Imidazolium-based ILs is well known due to
ammonium persulfate (APS) solution (10%(w/v)) as an initiator
their many fascinating properties which have been of special
and 15 mL (0.1 mmol) of tetramethylethylenediamine (TEMED) as
an activator were added to the solution. Prior to polymerization,
the reaction solution was purged with nitrogen for at least 30 min
to remove oxygen. Polymerizations were performed for 24 hours
at 80 ꢀC. The reaction solution became slightly yellowish and
viscous during the reaction. Aer completion of the reaction, all
possible impurities were removed by extraction with MC from the
importance in research elds such as surface wettability control,
catalyst molecule-supporting, nanostructure construction, and
green chemistry.32–37 The prepared copolymer (p-NIBIm) with
10 mol% concentration of the permanently cationic N-butyl-
imidazolium unit was appeared to have the LCST (lower critical
solution temperature) of 38.2 ꢀC and the surface charge of
+9.8 mV at pH ¼ 7. The temperature-dependent morphology
aqueous solution, following dialysis (membrane tubing, molec-
ular weight cutoff 12 000–14 000 Da, Spectrum Laboratories,
change and the temperature-dependent micelle size change also
were determined under dry and wet conditions, respectively, by
Savannah, GA, USA) against DW for 3 days and then freeze
SEM and zeta sizer. The ability to uptake and release a negatively
drying. To obtain the copolymer product as a pure solid mass, the
charged protein (BSA), respectively, below and above the LCST
polymer product clearly dissolved in 20 mL of cold DW was
incubated in 60 ꢀC for one hour and then the precipitated white
(25–45 ꢀC) were determined with comparing with that of
p-NIPAAm. The extraordinary protein-entrapping and releasing
sold was separated by centrifugation from the aqueous solution.
ability of the IL-doped copolymer will potentially be applied to be
The process was repeated three more times for further purica-
tion. Finally the puried polymer product dissolved in 20 mL of
cold DW was freeze dried and 1.38 g of the polymer product that
used as a novel delivery system for negatively charged molecules
such as a natural (or synthetic) drug and DNA.
looks like white cotton wool was obtained (about 96.5 wt% yield).
2 Materials and methods
2.1 Materials
2.4 Synthesis of poly(1-butyl-3-vinylimidazolium bromide),
p-BVIm, and poly(N-isopropylacrylamide), p-NIPAAm, as
N-Isopropylacrylamide (NIPAAm) was purchased from ACROS
(USA) and puried by recrystallization from hexanes (HPLC
grade, Sigma-Aldrich, USA) prior to use. N-Vinylimidazole
(NVIm, Sigma-Aldrich, USA), 1-bromobutane (Sigma-Aldrich,
USA), ammonium persulfate (APS, Sigma-Aldrich, USA),
N,N,N0,N0-tetramethylethylenediamine (TEMED, Sigma-Aldrich,
USA) were used as received. The Bio-Rad DC Protein Assay II kit
was supplied by BMS (Korea). All other reagents and solvents
used were analytical grade and used as received.
reference polymers
N-Vinyl imidazolium-based ionic liquid polymer, p-BVIm, and
poly(N-isopropylacrylamide), p-NIPAAm, were synthesized as
2.2 Synthesis of 1-butyl-3-vinyl imidazolium bromide,
[BVIm]Br, as an IL monomer
N-Vinyl imidazolium-based ionic liquid monomer was prepared
via a simple one-step quaternization reaction of N-vinyl imid-
azole with 1-bromobutane. Reaction mixture of 2.35 g (25
Scheme 1 Schematic illustration of the preparation of poly(NIPAAm-
mmol) 1-vinyl imidazole and 4.11 g (30 mmol) 1-bromobutane co-BVIm) or p-NIBIm.
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follows. 20 mmol of 1-butyl-3-vinylimidazolium bromide value is the average size and the distance between the two ends
([BVIm]Br) or N-isopropylacrylamide were dissolved in 20 mL of at the base is reected in the standard deviations. The
DW, and then 10 mL (0.5 mmol) of ammonium persulfate (APS) morphologies of micelles without and with BSA molecules in a
solution (10%(w/v)) as an initiator and 15 mL (0.1 mmol) of dry state were characterized by SEM (S-4300, HITACHI, Japan).
tetramethylethylenediamine (TEMED) as an activator were For this, the polymer solution (0.1 mg mLꢂ1) was dropped onto
added to the solution. Prior to polymerization, the reaction a cover glass and then dried at 25 ꢀC and 50 ꢀC in the dark
solution was purged with nitrogen for at least 30 min to remove overnight. The zeta (x) potential values were recorded using a
oxygen. Polymerizations were performed for one hour at room zeta sizer (ZEN 3600, Malvern) at room temperature as a func-
temperature. All possible impurities in the aqueous solution tion of pH (3–10) using a mixture of 0.1 M acetic acid and 0.2 M
were removed by dialysis (membrane tubing, molecular weight sodium acetate as buffering in water. The desired pH was
cutoff 12 000–14 000 Da, Spectrum Laboratories, Savannah, GA, adjusted by HCl or NaOH solutions, and pH values were
USA) against DW for 3 days. The obtained crude product of p- measured by a pH-meter (Orion 3 star, thermo scientic, Sin-
BVIm was puried by ash column chromatography (MC/ gapore). All the measurements were conducted at least in trip-
methanol) to give >90–80 wt% yield, while the pure p-NIPAAm licate for each experimental group and the average values were
was obtained by washing with methylene chloride several times. represented with the standard deviation.
2.9 Determination of colloidal stability of the p-NIBIm
solution in body temperature
2.5 Chemical characterization of the polymer
The chemical characterizations of the synthesized monomer
and polymer products were performed by FT-IR spectroscopy
Colloidal stability of the p-NIBIm solution was tested by Tur-
biScane LAb (Leanontech, a pulsed near infrared light source
(880 nm)). The copolymer solution (0.5 mg mLꢂ1) of 35 mm
1
(Nicolet 380, Thermo Fisher, USA) and H-NMR (Bruker, Ultra-
shield 400 PLUS, USA). FTIR spectra were taken using a KBr
window coated with the copolymer solution (5 wt%) in ethanol.
1H-NMR spectra of products were obtained at 400 MHz using d6-
dimethyl sulfoxide as solvent.
ꢀ
height was lengthwise scanned every 6 h for 72 h at 37 C. The
light backscattered by the sample solution (135ꢀ) (or trans-
mission) was measured.
2.10 Determination of the temperature-dependently loaded
and released BSA amount of p-NIBIm
2.6 Average molar mass determination of polymers
MALDI-TOF (matrix-assisted laser desorption ionization time of
ight) mass spectroscopy (Voyager-DE STR; Negative Polarity)
was successfully employed to determine average molar mass of
the cationic copolymer. 10 mg of the copolymer sample were
dissolved in 1 mL of water and mixed with a-cyano-4-hydrox-
ycinnamic acid (HCC) matrix solution at a ratio of 1 : 9 (v/v,
polymer : matrix). The MALDI-TOF mass spectra showed the
corresponding average molar masses of the copolymer.
The amount of BSA molecules that were temperature-depen-
dently loaded in the polymer micelles and released from the
micelles was determined by Bio-Rad DC assay. The Bio-Rad DC-
Protein assay is a colorimetric assay based on a modied Lowry
protein assay method following detergent solubilization. The
amount of BSA in solution samples was determined using a
calibration curve over the range of 0.20–1.0 mg mLꢂ1 of the
peptide solution. Three sample solutions were prepared by
combining 1.0 mL of the aqueous polymer solution (0.1 w/v%)
and 1.0 mL of aqueous BSA solution (1.0 w/v%) at 4 ꢀC. The
sample solutions were incubated in a water bath, respectively,
of 25, 36, and 42 ꢀC and then were ltrated through a PTFE
membrane lter (0.1 mm pore size, Advantec, Japan) equipped
with a syringe at each temperature. 100 mL of the each ltrate
solution was put in three new test tubes and 500 mL of reagent A0
(a mixture of 20 mL of reagent S and 1.0 mL of reagent A) and 4
mL of reagent B were added into each test tube, following vor-
texing immediately. Aer 15 min, the absorbance of the each
sample can be read using UV-vis spectrometer (1601PC, Shi-
madzu) at 750 nm. The experiments were conducted at least in
triplicate for each experimental group (at 25, 36, and 42 ꢀC) and
the average values were represented with the standard
deviation.
2.7 LCST determination of the copolymer
The thermal behaviors of the prepared polymers were studied
utilizing DSC (DSC 131 evo, setaram, France). An aqueous
solution of the sample polymer was prepared in a concentration
of 5.0 wt% in DW. It exhibited endothermic and an exothermic
peaks, respectively, in heating and cooling processes with a rate
ꢂ1
ꢀ
ꢀ
of 2 C min between 25 and 60 C.
2.8 Determination of mean size and zeta potential of
polymer micelles without and with BSA molecules
Mean sizes of polymer micelles without and with the guest
molecules (BSA) under wet condition were measured inꢀ a
ꢀ
heating process with an interval of 3 C between 25 and 45 C
using zeta sizer nano ZS90 (Malvern, France). 1.0 mL of aqueous
polymer solutions (0.01 wt%, prepared at 4 ꢀC) without and with
1.0 mg of BSA were prepared using a mixture of 0.1 M acetic acid
2.11 Cytotoxicity assay
and 0.2 M sodium acetate as buffering. Their size distribution The cytotoxicity on p-NIBIm micelles was determined by Cell
curves were obtained as a Gaussian type curve. The size distri- Counting Kit-8 (CCK-8) according to the manufacturer's
bution curves were almost symmetrical to the vertical line instructions (Dojindo Laboratories, Japan). The human
passing through the maximum, in all cases. The maximum embryonic kidney cell line, HEK 293, was purchased from
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American Type Culture Collection (ATCC, USA) and maintained important physicochemical properties of the thermo-sensitive
in Dulbecco's modied Eagle's medium (DMEM; Sigma, USA) polymer compared to those of the pure p-NIPAAm polymer.
containing 10% fetal bovine serum (FBS; Gibco, USA),
We determined the molar mass distribution of the IL-doped
100 U mLꢂ1 penicillin and 100 g mLꢂ1 streptomycin (Gibco, copolymer, p-NIBIm, using MALDI-TOF mass spectroscopy. The
USA) in a humidied incubator with about 90% and 5% CO2 at results were compared with those of p-NIPAAm and p-BVIm that
37 ꢀC. To test the cytotoxicity, 100 mL of cell suspensions of HEK were prepared as references, respectively, from NIPAAm and
293 cells were seeding at a density of 5 ꢃ 103 cells per well in the [BVIm]Br monomers in the same manner. As shown in Fig. 1
96-well microplates and pre-incubated overnight. Concentra- and S2,† the number- and weight-average molar masses (Mn and
tion sets of the p-NIBIm micelle solutions were prepared by a 2- Mw) of the copolymer appeared to be, respectively, around 1353
fold serial dilution with culture media from 8 mg mLꢂ1 to and 2001 g molꢂ1, showing that the molar mass distribution
0.03 mg mLꢂ1. Each set of 10 mL was added to each well (n ¼ 4) (Mw/Mn) is 1.47, while Mn and Mw of p-NIPAAm were, respec-
and continued to culture for 1–3 days. At each time point, tively, around 1998 and 2521 g molꢂ1, showing Mw/Mn ¼ 1.26.
the plate was further incubated for 2 hours in addition with For the case of p-BVIm, Mn and Mw were recorded as being
10 mL of CCK-8 solution to each well and then the optical around 2102 and 2114 g molꢂ1 (Mw/Mn ¼ 1.0).
density (OD) was measured at absorbance of 450 nm using a
To assess the presence of the two components, NIPAAm and
microplate reader (PerkinElmer, USA). The rates of the cell [BVIm]Br, within the copolymer, p-NIBIm, FT-IR (see Fig. S3†)
viability were calculated by the following equation: cell viability and 1H-NMR spectra (see Fig. 2 and S1†) were obtained and
(%) ¼ (ODp-NIBImO/ODcontrol) ꢃ 100%, where ODcontrol was compared to those of the homopolymers, p-NIPAAm and p-
obtained in the absence of polymers and ODp-NIBIm in the BVIm. As expected, the characteristic peaks of p-NIBIm were
presence of polymers.
observed in the FR-IR spectrum as follows: 3030–2874 cmꢂ1 (sp2
C–H stretching of BVIm units; sp3 C–H stretching of NIPAAm
and BVIm units), 1670–1580 cmꢂ1 (C]O stretching of NIPAAm
units and C]C and C]N stretching of BVIm units), 1535 cmꢂ1
(C(]O)–N–H bending of NIPAAm units), 1458 cmꢂ1 (CH2
bending of NIPAAm and BVIm units), 1366 cmꢂ1 (CH3 bending
3 Results and discussion
3.1 Synthesis and characterization of the IL-doped
copolymer, poly(NIPAAm-co-BVIm) or p-NIBIm
In this study, a permanently ionic and thermo-sensitive copol- of NIPAAm and BVIm units), and 1030–1300 cmꢂ1 (C–N
ymer with the relatively hydrophobic NIPAAm component as a stretching of NIPAAm and BVIm units). In addition to the
major part and the hydrophilic (or ionic) N-vinyl imidazolium characteristic peaks, a broad characteristic band of water
component (imidazolium-based ionic liquid (IL)) as a minor appeared at 3200–3600 cmꢂ1 for the p-NIBIm sample, denoting
part was designed and synthesized as a carrier, especially for the hydrophilic character of the copolymeric derivative. 1H-
negatively charged (or ionic or highly polar) guest molecules, NMR spectrum of the p-NIBIm copolymer also is given in Fig. 2
including synthetic drug, gene, and protein (Scheme 1). As and showed characteristic peaks that belong to the protons of
shown in Scheme 1, the chemical integration of the IL moiety NIPAAm and BVIm monomer units. To check the molar ratio of
into the p-NIPAAm polymer chain was achieved via copoly- both monomer units consisting of the copolymer chain, the
merization of 1-butyl-3-vinyl imidazolium bromide ([BVIm]Br) integrals of the broad signals (at 3.84 ppm for the isopropyl CH
with NIPAAm at the molar ratio of 1 to 10. Actually, controlling proton of NIPAAm unit and at 4.15 ppm for the N–CH2 proton of
the concentration of the positively charged (or hydrophilic) BVIm unit) were compared. In the spectrum, the integrals were,
imidazolium moiety within the copolymer chain could be respectively, 10 and 2, and these denote that the copolymer p-
expected to be an effective method for tuning the most impor- NIBIm contains about one BVIm unit per 10 NIPAAm units.
tant physicochemical and morphological properties of thermo-
sensitive drug-carriers, such as solubility in water, LCST,
surface charge, pH at IEP, micelle size, guest molecule-switch-
ing (or -attaching and -detaching) abilities at the suitable
temperatures, and the capacity for drug-carrying. Prior to the
copolymerization, N-vinyl imidazolium-based ionic liquid
monomer ([BVIm]Br; 1-butyl-3-vinylimidazolium bromide) was
prepared via a C–N coupling of N-vinylimidazole with 1-bro-
mobutane (see the 1H-NMR spectrum in Fig. S1 of the ESI†).
Then the copolymerization with NIPAAm monomer was
accomplished using 10 mol% of [BVIm]Br monomer to prepare
poly(NIPAAm-co-BVIm) or p-NIBIm. The optimal concentration
(10 mol%) of the cationic unit within the prepared copolymer
chain to show the most suitable LCST range (38–42 ꢀC) and an
excellent drug-carrying ability was determined by gradual
increasing of the IL monomer concentration from 0 up to
10 mol%, because, as mentioned above, the chemical doping of
the hydrophilic IL units could be considered to change the most Fig. 1 MALDI-TOF spectrum of the copolymer, p-NIBIm.
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Fig. 2 1H-NMR spectrum of the p-NIBIm copolymer.
1
Consequently, FT-IR and H-NMR spectra indicate an effective slightly different concentrations (distribution) of imidazolium
integration of BVIm monomers in the copolymerization with units within the copolymer molecules. Moreover, the repulsion
NIPAAm monomers.
between the cationic units (imidazolium units) of the copoly-
mer molecules consisting of a micelle also could be considered
to increase the phase transition temperature of the micelle,
subsequently resulting in shiing the LCST of the copolymer
micelle to the higher temperature than that of p-NIPAAm
micelle. Theoretically, doping of about 1.0 mol% of BVIm units
into p-NIPAAm appeared to increase by about 0.6 degree of
LCST. The obtained LCST value (38.2 ꢀC) of p-NIBIm may be
highly meaningful for in vivo applications, because the phase
transition temperature is between body temperature (37 ꢀC) and
hyperthermia temperature (42 ꢀC). This type of LCST shi also
can be found in the previous reports that thermo-sensitive
polymers with amine functional groups shied their LCSTs to
higher temperature by protonation under acidic surrounding
condition.21–25 However, unlike these, the p-NIBIm copolymer
with permanent cationic moieties was not affected by the acidic
pH environments (pH ¼ 4–6) and their LCST value also cannot
be changed (Fig. S4b†), demonstrating high applicability as an
efficient system to deliver drug exactly to the target site.
Consequently it can be considered that the introduction of the
permanently cationic moieties within a polymer chain is very
effective to increase both the loading efficacy and the trans-
ferring stability of drug molecules to the target site.
3.2 Thermal behavior of the IL-doped copolymer, p-NIBIm
The thermal behavior of the IL-doped copolymer, p-NIBIm,
was studied utilizing DSC and compared to those of the
homopolymers, p-NIPAAm and p-BVIm, synthesized as a refer-
ence. As well known and expected, an aqueous solution sample
(5 wt%) of p-NIPAAm exhibited an endothermic peak at 32.1 ꢀC
(the LCST) in the heating process and an exothermic peak at
ꢀ
29.0 C in the cooling process (Fig. 3a), while for the case of p-
BVIm any phase transition at the temperatures ranging from 5
to 60 ꢀC was not observed. However, the IL-doped copolymer
showed a clear phase transition and a good reversibility that was
conrmed via several continuous heating and cooling cycles
(Fig. 3b and S4a†). Fig. 3b shows that an endothermic peak of
the copolymer product in the heating process appeared at
ꢀ
38.2 C (the LCST), whereas an exothermic peak in the cooling
ꢀ
process occurred at 36.2 C. The temperature responsive prop-
erty of p-NIBIm will be caused by the coexistence of relatively
hydrophobic (or/and nonionic) and hydrophilic (or/and ionic)
parts within the IL-doped copolymer. It is thought that the
relatively broad peaks for p-NIBIm may be associated with the
3.3 Surface charge of the partially ionic copolymer, p-NIBIm
Zeta (x) potential values of p-NIPAAm, p-NIBIm, and p-BVIm
were measured as a function of pH (3–10) in a buffering system
of 0.1 M acetic acid and 0.2 M sodium acetate at room
temperature with the help of zeta sizer to conrm the cationic
character of the IL-doped p-NIBIm copolymer. As shown in
Fig. 4, initially the nonionic polymer, p-NIPAAm, showed an
isoelectric point (IEP) at pH ¼ 8.6 and positive zeta (x) potential
values below the IEP (a maximum value of about 11.5 mV at
pH ¼ 4), indicating that the polymer chain (and it's LCST)
consisting of amide functional groups is pH-dependant and
Fig. 3 LCST determination of (a) p-NIPAAm and (b) IL-doped p-NIBIm
using DSC scan.
26742 | RSC Adv., 2014, 4, 26738–26747
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weakly positive under neutral condition. However, in the case of
the IL-doped copolymer, p-NIBIm, no IEP appeared at the range
of pH ¼ 4–10, indicating that it has permanently positive
charges, regardless of the external pH change. The zeta (x)
potential change of p-NIBIm at the range of pH ¼ 4–10,
however, appeared to be only about 8 mV, meaning that only 10
mol% of the cationic BVIm moieties are the main factor that
affect the charge and the LCST of copolymer micelles. These
results will be caused by the coexistence of the pH-indepen-
dently cationic BVIm moieties and weakly pH-dependent
NIPAAm moieties. Only the NIPAAm moieties responded to the
protonation or deprotonation process slightly affecting the
charge and the LCST of copolymer micelles. Additionally, Fig. 4
demonstrates that the poly-cationic IL-polymer consisting of
100% BVIm units, p-BVIm, showed an extremely high zeta (x)
potential value, about 32.5 mV, at pH ¼ 7 and the pH-inde-
pendent behavior of zeta (x) potential values at the range of
pH ¼ 4–10. The three different polymers with increasing
concentration of BVIm unit exhibited increasing zeta (x)
potential values at pH ¼ 7, for example, +0.3 mV of p-NIPAAm,
+9.8 mV of p-NIBIm, and +32.5 mV of p-BVIm.
3.4 Temperature-dependant morphology change of the IL-
doped copolymer micelles, p-NIBIm, and their complexes with
BSA, p-NIBIm/BSA
Temperature-dependant morphology change of p-NIBIm
micelles in an aqueous solution was tested by scanning electron
microscope (SEM). The sample was prepared on a glass by
drying a drop of the aqueous sample (0.01 wt%) at 25 (<the
LCST) and 50 (>the LCST) ꢀC. As shown in Fig. 5a and b, the SEM
images demonstrate that the brous bundle-like aggregates
(about <3 mm in length) were found for the sample dried at
25 ꢀC. The copolymer molecules perhaps existed as hydrated
random coils or swelled globules at the lower temperature and
then the coils or swelled globules might gather or collapse to the
brous bundle-like aggregates during the drying process. When
ꢀ
the drying temperature increased to 50 C, the brous bundle-
like morphology was dramatically changed to compact globules
of about <500 nm in diameter. When looking in depth at the
SEM image we can also discover that the compact globule is an
aggregate of several smaller globules together of about <200 nm
in diameter.
We also tested the morphology change aer their complex
formation with BSA by SEM. For this, 1.0 mL of aqueous poly-
mer–BSA complex solution (0.011 wt%) containing 0.01 mg of
The colloidal stability of the p-NIBIm copolymer solution
(0.5 mg mLꢂ1) was tested by Terbiscan LAb. For this, the
ꢀ
aqueous solution was scanned 12 times at 37 C for 72 hours.
ꢀ
polymer and 0.1 mg of BSA were used. Aer drying at 25 C, a
These were conrmed by measuring the backscattered light (or
transmission) of a pulsed near infrared light source of 880 nm
wavelength (see Fig. S5†). During the entire scanning time, any
noticeable change of the light uxes backscattered by the
sample solution was not detected, demonstrating highly stable
and uniform dispersion of p-NIBIm copolymer micelles in the
solution. Here the uctuations of the light uxes occurred
during the latter scanning periods were caused by air bubbles
dramatic morphology change from the brous bundle-like
aggregates (about <3 mm in length) to the circular aggregates
(about <1.5 mm in diameter) and aggregates of BSA molecules
encapsulated inside the circular aggregates were observed (see,
respectively, the red- and blue-colored arrows in Fig. 2c). Here, it
should be notable that the circular aggregates have loose
spaces, meaning they existed as swelled and hydrated globules
encapsulating large amount of BSA molecules under wet
ꢀ
formed within the closed system of 37 C.
ꢀ
conditions at 25 C. As shown in Fig. 2d, the compact globule-
Fig. 5 SEM microscopic images of the p-NIBIm copolymer and the p-
NIBIm/BSA complex micelles: (a and b) p-NIBIm samples prepared by
drying, respectively, at 25 and 50 ꢀC; (c and d) p-NIBIm/BSA complex
samples prepared by drying, respectively, at 25 and 50 ꢀC (red circle
Fig. 4 The zeta (x) potential values as a function of pH in 0.1 M acetic and arrow: swelled and deswelled p-NIBIm micelle, blue arrow: BSA
acid and 0.2 M sodium acetate.
aggregate).
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like aggregates again appeared at the higher drying-temperature ꢁ 5.1 to 39.8 ꢁ 3.6 nm) under the same temperature condition
of 50 ꢀC and their mean size also returned to the size of free p- and a rapid deswelling around the LCST (32–34 ꢀC) with a little
ꢀ
NIBIm micelles without BSA (about <200 nm in diameter). initial reduction below 31 C (see Fig. S6†). The copolymer p-
Moreover, a large scale of BSA aggregates that were released via NIBIm with permanent positive charge clearly revealed the
phase transition and size contraction during the heating following interesting properties compared to the p-NIPAAm: the
process from the swelled globules encapsulating a large quan- about 6 degree higher LCST, the 1.4–2.3 fold larger micelle size
tity of BSA also was found. Consequently, the SEM results in diameter in the entire region of the tested temperatures, and
comprehensively illustrate that the morphology and size the about 2.7 fold larger volume contraction in cm3. As
changes should be clearly related to the temperature-dependant mentioned above, the molar masses (Mn, Mw) of p-NIBIm were
BSA-loading and -releasing behaviors of p-NIBIm micelles, smaller than those of p-NIPAAm. Nonetheless, there is no doubt
which occurred via the thermo-responsive phase transition of p- that the increase of the micelle diameter can be caused by the
NIBIm micelles from swelled globules to desolvated compact strong repulsive force between positive charged imidazolium
ꢀ
globules in the temperature range (25–50 C).
rings inside the copolymer micelles. Moreover, it also should be
noted that the level of the micelle contraction for the IL-doped
copolymer (p-NIBIm) at the tested temperature range (25–45 ꢀC)
is much higher than the level of the nonionic polymer (p-
NIPAAm). All these results may indicate that the p-NIBIm
copolymer, which may exist as swelled micelles below the LCST,
can be expected to show a high adsorption efficacy of negatively
charged molecules including drugs, genes and proteins owing
to the charge–charge interaction between the positively charged
host molecules and negatively charged guest molecules. More-
over, the high level of the micelle contraction at the LCST also
may indicate an efficient releasing of the entrapped guest
molecules via the thermo-responsive phase transition from a
swelled globule to a desolvated compact globule.
The next step in this research is to test whether the IL-doped
p-NIBIm micelles will function as planned, for example, to
encapsulate and to release the protein BSA as a negatively
charged model molecule, respectively, below and above the
LCST. For this, the temperature dependent micelle size change
of the aqueous p-NIBIm sample containing the protein BSA was
tested in the temperature range of 25–45 ꢀC using zeta sizer (see
3.5 Temperature-dependant size change of the IL-doped
copolymer micelles, p-NIBIm, and their complexes with BSA,
p-NIBIm/BSA
The size (or volume) change of p-NIBIm micelle and p-NIBIm/
BSA complex micelle, depending on the temperature change
ranging from 25 to 45 C, was tested under wet conditions by
ꢀ
zeta sizer. For this, aqueous samples (0.1 wt%) of the IL-doped
copolymer, p-NIBIm, without and with BSA (1.0 wt%) were
subjected to the instrument. As summarized in Fig. 6, the
micelle volume of the pure p-NIBIm sample was reduced by
about 8.09 ꢃ 10ꢂ15 cm3 (decrease in diameter from 253 ꢁ 12.1
to 90.5 ꢁ 7.8 nm) with increasing temperature from 25 to 45 ꢀC,
showing a slow reduction between 25–37 ꢀC and a rapid
contraction around the LCST (38–39 ꢀC). The size decrease
obviously is caused by the phase transition of swelled globules
and their volume contraction to compact globules. In contrast,
the nonionic polymer, p-NIPAAm, showed the volume contrac-
tion of only 3.05 ꢃ 10ꢂ15 cm3 (decrease in diameter from 180.6
Fig. 6 The temperature-dependent micelle size changes of p-NIBIm copolymer under wet conditions.
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Fig. 6) and compared to that of p-NIPAAm (see Fig. S6†). 1.0 mL
of the aqueous complex solution containing 0.1 mg of the
polymer, p-NIBIm or p-NIPAAm, and 1.0 mg of BSA were sub-
jected to the instrument. As shown in the gures, the sizes of p-
NIBIm/BSA and p-NIPAAm/BSA complex micelles at 25 ꢀC
appeared to be, respectively, 491.0 ꢁ 7.2 nm and 332.0 ꢁ 9.8 nm
in diameter, meaning the volumes of, respectively, about 62.0 ꢃ
10ꢂ15 cm3 and 19.1 ꢃ 10ꢂ15 cm3. When the temperature further
increases over the respective LCSTs, p-NIBIm/BSA complex
micelles were reduced by about 61.9 ꢃ 10ꢂ15 cm3 in volume
ꢀ
(from 491.0 ꢁ 7.2 nm in diameter at 25 C to 55 ꢁ 6.5 nm in
diameter at 45 ꢀC), showing a slow reduction between 25-37 ꢀC
and a rapid contraction around the LCST (37–42 ꢀC), whereas p-
NIPAAm/BSA complex micelles exhibited a reduction of about
18.7 ꢃ 10ꢂ15 cm3 in volume (from 332 ꢁ 9.8 nm in diameter at
25 ꢀC to 89 ꢁ 5.7 nm in diameter at 45 ꢀC), showing a slow and
Fig.
7
The different BSA-loading capacities of p-NIBIm and p-
NIPAAm at 25 ꢀC: (a) the initial BSA concentration, (b) the filtrate
obtained from the p-NIBIm/BSA solution at 25 ꢀC and (c) the filtrate
obtained from the p-NIPAAm/BSA solution at 25 ꢀC.
ꢀ
consistent reduction between 25–39 C. Here it is very impres-
sive that p-NIBIm/BSA complex micelles were intensively con-
tracted in a certain range (38–42 ꢀC) around the LCST, while p-
NIPAAm/BSA complex micelles were continually contracted over
the entire range of 25–45 ꢀC. The about 3.3 times larger volume
and volume contraction of p-NIBIm/BSA complex micelles than
p-NIPAAm/BSA complex micelles may reect the higher capacity
to load and release drugs.
ꢀ
The BSA concentrations remained in micelles of 37 C and
further released from the micelles between 38–42 ꢀC were taken,
respectively, as the loaded and released BSA amounts especially
for the p-NIBIm polymer sample. Loading and releasing tests of
the BSA molecules for p-NIBIm, therefore, were accomplished
through elevating the temperature of the sample solution
prepared at 4 ꢀC to body temperature (37 ꢀC) and clinical
hyperthermic temperature (42 ꢀC). Fig. 8 shows BSA-loading
capacity at 37 ꢀC and BSA-releasing capacity at 38–42 ꢀC for the
p-NIBIm micelles. When the temperature of two p-NIBIm/BSA
solution samples increased, respectively, to 37 and 42 ꢀC, as
shown in Fig. 8, respectively, 1.4 mg and 0.38 mg of BSA were
le in the copolymer micelles. This means that 1.0 mg of p-
NIBIm can load 1.4 mg of BSA at body temperature and extrude
3.6 Temperature-dependant BSA loading and releasing
behaviors of the IL-doped copolymer, p-NIBIm
As the nal step for this research, the quantitative determina-
tions of the loaded and released BSA concentrations were
accomplished by Bio-Rad DC-Protein assay. For this, three
aqueous samples (2 mL) containing 1.0 mg of the thermo-
sensitive polymer (p-NIBIm or p-NIPAAm) and 10 mg of BSA
molecule were prepared at 4 ꢀC and then the temperature of the
three samples was slowly increased, respectively, to 25 (room
temperature), 37 (body temperature), and 42 ꢀC (clinical
hyperthermic temperature). Each sample solution was rapidly
ltered through a syringe lter equipped with PTFE membrane
lter (0.1 mm pore size, Advantec, Japan) at each temperature,
following adding the DC protein assay reagent to each ltrate
ꢀ
1.02 mg (72.9%) of the in the micelle of 37 C remaining BSA
amount via a deswelling process of the swelled copolymer
micelles between 38–42 ꢀC, while p-NIPAAm micelles can
and measuring the UV/Vis absorbance of the ltrate at lmax
¼
750 nm. The BSA concentrations in the ltrate and encapsu-
lated in polymer micelles were calculated via a standard curve,
created by plotting the known BSA concentration on x axis and
the absorbance of BSA/DC complexes at lmax ¼ 750 nm on y axis
(Fig. S7†). Fig. 7 shows different BSA-loading capacities of the p-
ꢀ
NIBIm and the p-NIPAAm samples at 25 C. The BSA amount
encapsulated in 1.0 mg of p-NIBIm polymer appeared to be
about 5 times larger than that of p-NIPAAm, for example,
3.28 mg for p-NIBIm and 0.64 mg for p-NIPAAm. These quan-
titative results interestingly are consistent with the results of
micelle volume increase obtained by zeta sizer at 25 ꢀC (see
Fig. 6). The higher BSA adsorption ability of p-NIBIm micelles is
denitely associated with the charge–charge interaction
between negatively charged BSA molecules and positively
charged imidazolium rings within the swelled micelles of the
copolymer chains.
Fig. 8 The BSA-loading and -releasing capacities of the p-NIBIm
copolymer, respectively, at 37 ꢀC and 38–42 ꢀC: (a) the initial BSA
concentration (5 mg mLꢂ1), (b) filtrate obtained at body temperature
(37 ꢀC) and (c) filtrate obtained at 42 ꢀC.
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ꢁ 7.8 nm in diameter (about 95.4% volume contraction). The
thermo-sensitive behaviors to entrap BSA protein at body
temperature (37 ꢀC) and to release the protein between 38–42 ꢀC
(near the LCST) also were tested by sizing the complexes of p-
NIBIm/BSA using zeta sizer, by SEM, and also by colorimetric
assay (Bio-Rad DC Protein Assay), resulting in a maximum
entrapment of 1.4 mg BSA (about 140% loading) for 1.0 mg of
the polymer at body temperature (37 ꢀC) and in a maximum
release of 1.02 mg BSA for 1.0 mg of the polymer (about 73%
release of the entrapped amount) at the temperature range of
ꢀ
Fig. 9 Viability of HEK 293 cells incubated with p-NIBIm micelles in 38–42 C. The in vitro toxicity of the p-NIBIm micelles without
the concentration range of 0–8 mg mLꢂ1 for 1 day and 3 days at 37 ꢀC.
drug for human embryonic kidney (HEK 293) cells was minimal
in the range of <0.125 mg mLꢂ1. These results revealed IL-doped
and temperature responsive co-polymeric systems have high
applicability as a novel delivery system for negatively charged
molecules as a natural (or synthetic) drug and DNA.
Data were shown as mean ꢁ S.D. (n ¼ 4).
ꢀ
encapsulate only 0.64 mg at 25 C and extrude almost all the
BSA molecules below body temperature. In conclusion, the IL-
doped copolymer p-NIBIm, unlike the p-NIPAAm, are consid-
ered to be potentially useful as a smart delivery system of
negatively charged molecules, such as BSA protein, because p-
NIBIm can carry up to about 1.0 mg drug per 1.0 mg of the
polymer to the target site between body temperature and
hyperthermic temperature.
Acknowledgements
This study was supported by the research fund of Dankook
University in 2012.
Notes and references
3.7 Cytotoxicity assay
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