Ultrasensitive (Co)polymers based on poly(methacrylamide) structure with fining-tunable pH responsive value

Article abstract of DOI:10.3390/molecules23081870

Novel pH responsive copolymers with tertiary amine groups were prepared by free radical polymerization with 2-(dialkylamino)ethyl methacrylate monomers. These polymers were pH sensitive with the ability to be responsively fine-tuned in aqueous solution, which was proven through titration, transmittance measurements, and proton nuclear magnetic resonance spectroscopy. The polymers were soluble in water at low pH values, induced by electrostatic repulsion between amine groups, and aggregated above their pK_a value due to the hydrophobic effect of the alkyls. The pH responsive values were precisely tuned from 7.4 to 4.8 by increasing the hydrophobic monomer ratio. Our work provides a novel approach for the development of ultrasensitive pH-responsive polymers for application in biomedical materials.

Full text of DOI:10.3390/molecules23081870

molecules
Article
Ultrasensitive (Co)polymers Based on
Poly(methacrylamide) Structure with
Fining-Tunable pH Responsive Value
Haiming Fan 1 , Po Li , Wei Li , Hui Li and Xiaonan Huang 2,*
ID
2
2
1
1
Shandong Provincial Key Laboratory of Oilfield Chemistry, School of Petroleum Engineering,
China University of Petroleum (East China), Qingdao 266580, China; Haimingfan@126.com (H.F.);
lihui0714_upc@163.com (H.L.)
Department of Chemistry, Capital Normal University, 105 West 3rd Ring North Rd, Beijing 100048, China;
2
2
150702055@cnu.edu.cn (P.L.); 2120702055@cnu.edu.cn (W.L.)
*
Correspondence: huangxn@cnu.edu.cn
Academic Editors: Jianxun Ding, Yang Li and Mingqiang Li
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 25 June 2018; Accepted: 15 July 2018; Published: 27 July 2018
Abstract: Novel pH responsive copolymers with tertiary amine groups were prepared by free
radical polymerization with 2-(dialkylamino)ethyl methacrylate monomers. These polymers were
pH sensitive with the ability to be responsively fine-tuned in aqueous solution, which was proven
through titration, transmittance measurements, and proton nuclear magnetic resonance spectroscopy.
The polymers were soluble in water at low pH values, induced by electrostatic repulsion between
a
amine groups, and aggregated above their pK value due to the hydrophobic effect of the alkyls.
The pH responsive values were precisely tuned from 7.4 to 4.8 by increasing the hydrophobic
monomer ratio. Our work provides a novel approach for the development of ultrasensitive
pH-responsive polymers for application in biomedical materials.
Keywords: fine-tuning; pH responsive; poly(methacrylamide)s; phase transition
1
. Introduction
The development of tumor-targeted drug delivery systems have attracted increasing interest
given their potential to address therapeutic issues in clinical practice [
focused on designing and improving specific transport and activation of intelligent materials that
have stimuli responsive properties [ 13]. An interesting aspect of the physiology of tumors is their
1–7]. Many studies have
8–
lower extra-cellular pH (6.5–7.2) compared to the surrounding normal tissues and blood, thus pH
can be considered as an ideal trigger for tumor tissues and tumor cells [14]. To target the acidic
bio-compartments, several smart materials with pH-responsive properties were designed to increase
target efficacy. For instance, the Kobayashi group developed a small molecule system to target the
tissue of the mice. This pH of this system can transition across two values of pH value, which
means a 200-fold proton concentration change [15–17]. Despite these remarkable advances, selective
targeting by pH-sensitive materials of different endocytic components is challenging owing to the
small pH differences in these compartments, such as early endosomes (5.9–6.2) versus lysosomes
(
5.0–5.5) [18–20].
Various pH-responsive polymer systems have been studied, such as liposomes [21], micelles [22
nanoparticles [24], and nanogels [25 27]. The pH-responsive polymers can undergo phase transition
at a specific pH values and direct spontaneous assemblies [28 31]. Polyelectrolytes, which
,23],
–
–
contain weak acidic or basic groups, have been exploited as pH responsive polymers, inspiring
advances in polymer research. Polybases, like poly(2-(diethylamino)ethyl methacrylate) (PDEAEMA),
Molecules 2018, 23, 1870; doi:10.3390/molecules23081870
www.mdpi.com/journal/molecules

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poly(4 or 2-vinylpyridine) (PVP), and poly(vinyl imidazole), were developed for biomedical
applications in gene transfer, bio-imaging, and controlled drug release, due to their excellent
physicochemical properties [32
chains can accept protons under acidic condition and release them in basic environments to induce
phase transition [35 36]. For instance, PDEAEMA dissolves in acidic aqueous solution due to the
–34]. The amine group, pyridine, and imidazole group in polymer
,
electrostatic repulsions between the protonated amine groups, but undergoes a precipitation above
pH 7.5, caused by the hydrophobic effect of substituted ethyl group. However, a challenging issue
faced by these widely used polybases is that their phase transition values from the solution state to the
precipitation state are usually fixed. Therefore, they are less accurate when targeting cell organelles
with different pH microenvironments. The broad pH transition range of these polybases also limits
the increase in their responsive property. To develop responsive polymers with narrow transition
pH ranges, Armes et al. [37] reported a novel polybase system, poly(2-(diisopropylamino)ethyl
methacrylate) (PDPAEMA), with a phase transition at pH 6.3 and a sharp pH transition, providing a
method by which to prepare ultra-sensitive pH-responsive polymers.
In this work, we developed a series of novel reversible pH-responsive (co)polymers with
a fine-tunable pH transition value. These polymers were synthesized using a free radical
polymerization method with tertiary amine-based monomers, 2-(dibutylamino)ethyl methacrylate
and 2-(dimethylamino)ethyl methacrylate. At low pH, the (co)polymers were in the unimer state
due to the electrostatic interaction and hydrophilicity of the protonated tertiary amine units. At high
pH, the stronger hydrophobicity of the alkyl groups on the deprotonated amines led to (co)polymer
aggregation. All the (co)polymers showed rapid phase separation at their pKa, and the pH transition
range can be controlled within a very narrowly defined value.
2
. Results
2
.1. Synthesis of the (Co)polymers
A series of random copolymers poly(2-(dimethylamino)ethyl methacrylate)-co-2-(dibutylamino)
ethyl methacrylate (P(DMAEMA-co-DBAEMA)) were obtained by conventional free radical
polymerization with varying feed ratios of the monomers containing different tertiary amine groups.
Comparing the signal integrals of the proton peaks at 2.3 ppm and at 2.5 ppm (Figure 1, Figures S1–S4),
the compositions of the copolymers could be determined, as summarized in Table 1. The molar ratios
of the monomer units in the copolymers were relatively close to the feed ratios. Due to the methacrylic
acid ester structure of the two monomers, random sequences of two monomer units were expected in
the copolymer chains.
1
Figure 1. Proton nuclear magnetic resonance ( H-NMR) spectrum of poly(2-(dimethylamino)ethyl
methacrylate)-co-2-(dibutylamino)ethyl methacrylate (P(DMAEMA0.73-co-DBAEMA0.27)) in CDCl3.

Molecules 2018, 23, 1870
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Table 1. Characterization of (co)polymers.
DMA Content
Mn b
(×10 ) (×10 )
Mw b
Yield
%)
pKa c
Copolymer
PDI
In Polymer a
(
4
4
In Feed
PDMAEMA
100
75
50
25
0
100
73
51
29
0
73
67
62
65
56
1.21
1.06
1.01
1.13
1.85
1.47
1.84
2.03
1.53
1.39
1.82
1.80
1.85
7.4
6.8
6.3
5.5
4.8
P(DMAEMA0.73-co-DBAEMA0.27
P(DMAEMA0.51-co-DBAEMA0.49
P(DMAEMA0.29-co-DBAEMA0.71
PDBAEMA
)
)
)
1.40
2.59
a
1
b
Percent molar ratio determined by hydrogen nuclear magnetic resonance ( H-NMR); Determined by gel
permeation chromatography (GPC) in tetrahydrofuran (THF) with polystyrene standards; c Determined by titration
during the heating process.
2
.2. pH Titration of Different Copolymers and Measurement of pKa
a
For pH-responsive polymers, the pK value is a critical parameter that can be evaluated through
titration experiments. Figure 2A shows the potentiometric titration curves for 0.5 mg/mL (co)polymer
solutions. NaOH solution was added to neutralize the protons. Potentiometric titration curves were
produced for (co)polymers by plotting the solution pH against the volume of added NaOH. Starting
from pH ~3, the addition of small aliquots of NaOH would neutralize the free protons and increase
the solution pH. The pH value of PDMAEMA increased continuously with higher NaOH content,
whereas for the other (co)polymers, especially PDBAEMA, titration curves were obtained with a
plateau at the polymer buffering region. This indicates that the added NaOH was consumed during
the deprotonation of the tertiary amine groups on the polymer pendent chain, which corresponds
to the phase transition of the polymer. With the completion of the process, further addition of base
increased the solution pH rapidly. For each sample, the pK
between the two equivalence points in the titration curve. With increasing DBAEMA monomer
amounts from 0% to 100%, as shown in Figure 2B, the average pK values of the polymers decreased
from 7.4 to 4.8, which was attributed to the hydrophobic property of butyl on amine. An approximately
linear relationship was observed between the pK values and DBAEMA monomer molar ratios of the
co)polymers, which indicates that the pK of the pH sensitive polycations can be controlled easily and
precisely by adjusting the hydrophobic properties of the pendent group.
a
values were calculated as the pH halfway
a
a
(
a
Figure 2.
(
A
) pH titration curves of (co)polymers. The volumes of NaOH (VNaOH) were normalized to
) pKa values of the tertiary amine-based polymers as a
the initial amount of amine ([R N] in mmol); (
B
3
0
function of DBAEMA ratio.

Molecules 2018, 23, 1870
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2
.3. Phase Transition Behaviors of the Fine-Tuning pH Responsive Polymers
The tenability of the (co)polymers’ pH-responsive properties were evaluated by transmittance
experiments. Figure 3A,B show the optical images and transmittance changes of the polymer solutions
as NaOH was added from initial acid solutions, where all of the polymers were completely dissolved.
Over the entire pH range in the measurements, the PDMAEMA solution remained clear with a
transmittance around 100%, which was likely due to the hydrophilicity of the dimethyl amino groups,
in good agreement with the titration experiment. However, with the incorporation of DBAEMA,
the turbidity of the polymer solutions increased sharply at critical pH values due to the aggregation of
the polymer chains. The phase transition pH range decreased with the increasing ratio of DBAEMA.
Compared to conventional pH responsive materials, the DBAEMA-based polymers displayed a much
sharper pH-dependent phase transition, where the transmittance from 100% to 0% was limited to
0.3 pH, indicating that only double the proton concentration could lead to complete turbidity of
the copolymers in aqueous solutions. Furthermore, the reversibility of the pH-responsive tertiary
amine-based polymers was confirmed by the PDBAEMA transmittance measurement. We also
analyzed the polymer solution during cyclic tests between pH 4.0 and pH 7.4. As shown in Figure 3C,
the dissolution and aggregation of the polymer solutions are fully reversible for at least five cycles,
which indicates the novel pH sensitive materials can be used repeatedly.
Figure 3.
(
A) Optical images of (co)polymer solutions at different pH. (B) The transmittance curves
of (co)polymer solutions with increasing pH values. (C) The pH reversibility study of PDBAEMA
solution with cycles between pH 4.0 and pH 7.4.
1
Proton nuclear magnetic resonance spectroscopy ( H-NMR) was also employed to investigate
the effect of pH on the phase transition behaviors of the polymers in aqueous solution. PDBAEMA
and P(DMAEMA0.73-co-DBAEMA0.27) were investigated as a function of pH in order to compare
the aggregation behavior of the polymers with different tertiary amine groups. 1,4-Dioxane, with
a single peak at 3.7 ppm, was used as the internal standard, as its intensity remained almost the
same before and after the polymer underwent a phase transition. The spectra of the PDBAEMA and
P(DMAEMA0.73-co-DBAEMA0.27) were recorded in the pH range of 4.1 to 8.5, and Figure 4 shows the
1
H-NMR spectra of the polymers in D O with the tertiary amines in different ionization states. At pH
2
4
.1 and 5.3, the amine groups were protonated; both PDBAEMA and P(DMAEMA0.73-co-DBAEMA0.27
were dissolved in the deuterated solution and proton resonance peaks for the segments of each
polymer were easily visualized. However, as pH increased from 5.5 to 6.6, the signal intensities
)

Molecules 2018, 23, 1870
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of the PDBAEMA dramatically decreased and disappeared at pH 7.8, when PDBAEMA became
completely deprotonated. The suppressed resonances of the polymer were due to the limitation of
the motion of the polymer chain, which can be attributed to the phase transition behavior driven
by the hydrophobic effect. For P(DMAEMA0.73-co-DBAEMA0.27), although the signal integrations of
the polymer signals decreased gradually when the pH increased above 6.3, the peaks of DMAEMA
segments were still observable even at pH 8.5. This drastic difference between PDBAEMA and
P(DMAEMA0.73-co-DBAEMA0.27) spectra can be attributed to the dehydration behaviors of the
polymers upon neutralization. The hydrophobic effect involving the butyl groups on the PDBAEMA
pendent segments enabled the polymer chains to pack densely in aqueous solution, thus leading to
more efficient dehydration and a sharper phase transition.
Figure 4. 1H-NMR spectra of (
A) P(DMAEMA0.73-co-DBAEMA0.27) and (B) PDBAEMA at different
pH in D O.
2
3
. Materials and Methods
3
.1. Materials
2
-(dibutylamino) ethanol was purchased from Alfa Aesar Company (Haverhill, MA, USA) and
2-(dimethylamino)ethyl methacrylate (DMAEMA) was purchased from Sigma-Aldrich (St. Louis,
MO, USA). CDCl , D O, and 1,4-Dioxane were purchased from Acros Co. (Beijing, China) and used
3
2
as received. Tetrahydrofuran (THF) was freshly purified by distillation over sodium prior to use.

Molecules 2018, 23, 1870
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2,2-Azoisobutyronitrile (AIBN) was recrystallized three times from methanol. Other solvents and
reagents were purchased from Beijing Chemical Reagent Co. (Beijing, China) and used as received.
3
.2. Synthesis of the DBA Monomer
The tertiary amine-based methacrylate monomer 2-(Dibutylamino)ethyl methacrylate (DBAEMA)
was synthesized by following a previously reported method [38]. 2-(dibutylamino)ethyl ethanol (17.3 g,
.1 mol), triethylamine (10.1 g, 0.1 mol), and inhibitor hydroquinone (0.11 g, 0.001 mol) were dissolved
in 100 mL THF and placed in a three-neck flask. To this solution, methacryloyl chloride (10.4 g,
0
0
2
.1 mol) was added dropwise with constant stirring. The resulting solution was refluxed in THF for
h and then filtered to remove the precipitated white triethylamine-HCl salts with completion of the
reaction. After removing THF solvent by rotary evaporator, the resulting residue was distilled in vacuo
◦
(
83–87 C at 0.05 mm Hg) as a colorless liquid. The obtained DBAEMA monomer was characterized
1
1
by H-NMR. H-NMR (TMS, CDCl , ppm): 6.09 (br, 1H, CHH=C(CH )-), 5.55 (br, 1H, CHH=C(CH )-),
3
3
3
4
.19 (t, J = 6.3 Hz, 2H, -OCH CH N-), 2.73 (t, J = 6.3 Hz, 2H, -OCH CH N-), 2.46 (t, J = 7.6 Hz, 2H,
2 2 2 2
-N(CH CH CH CH ) ), 1.93 (s, 3H, CH =C(CH )-), 1.41 (m, 4H, -N(CH CH CH CH ) ), 1.29 (m, 4H,
2 2 2 3 2 2 3 2 2 2 3 2
-
N(CH CH CH CH ) ), 0.89 (t, J = 7.3 Hz, 6H, -N(CH CH CH CH ) ), Yield: 56%.
2
2
2
3 2
2
2
2
3 2
3
.3. Synthesis of the (Co)polymers
The P(DMAEMA-co-DBAEMA) copolymers, PDMAEMA, and PDBAEMA were prepared by free
radical polymerization in THF with varying DBAEMA to DMAEMA molar feed ratios of 0:4, 1:3, 2:2, 3:1,
and 4:0, respectively. Typical procedures employed for the polymerization were as follows. Monomers
were dissolved in THF at a total concentration of 0.10 g/mL, to which azodiisobutyronitrile (AIBN)
(1.0 mol% relative to monomers) was added as a free radical initiator. After three cycles of freeze-thaw
to thoroughly remove oxygen, the tube was sealed under reduced pressure and the polymerization
◦
was performed at 60 C for 24 h. The polymers were purified by precipitation from diethyl ether twice,
collected by filtration, and dried under vacuum to obtain white powders. The molecular weights and
the composition of the (co)polymers were determined by gel permeation chromatography (GPC) and
1
H-NMR, respectively.
3
.4. Characterization of the (Co)polymers
1
H-NMR spectra of the monomers and (co)polymers were recorded in CDCl on a Varian-600
3
MHz spectrometer with tetramethylsilane (TMS) as the internal reference. The molecular weight
and molecular weight distributions of the (co)polymers were measured with gel permeation
chromatography (GPC) equipment consisting of WGE3010 pump, 3010 refractive index detector,
◦
and WGE Styra gel columns. The temperature of the columns was 35 C and THF was used as an
eluent at a flow rate of 1 mL/min. A series of narrowly dispersed polystyrene samples were used as
standards and Millennium 32 software was used to calculate the molecular weight and polydispersity.
3
.5. pH Titration
The pK values of different (co)polymers were detected by pH titration. When the prepared
polymer was dissolved in 0.1 N HCl to reach a final concentration of 5–10 mg/mL, the pH titration
experiment was performed by adding small volumes (50–100 L increments) of 0.1 N NaOH solution
a
µ
under stirring. The pH values of the solution were measured continuously using a Sartorius (Germany)
pH meter with a microelectrode.
3
.6. Turbidity Measurements by UV/Vis Spectroscopy
The pH-dependent phase transition of the polymers was determined by the turbidity of the
polymer solutions as a function of pH. The transmittance of the 1 mg/mL (co)polymer solutions
in 0.1 N HCl were measured at 500 nm through a 1 cm quartz cell on a Shimadzu 2550 ultraviolet

Molecules 2018, 23, 1870
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(
UV)-vis spectrometer. To adjust the pH, 0.2 N NaOH was added, and the final volumes of the polymer
solutions increased about 10% compared with initial volumes. The pH values were monitored with a
digital internal pH meter. Polymer-free deionized (DI) water was used as a reference.
1
3
.7. H-NMR Measurements of pH-Dependent Phase Transition
P(DMAEMA0.73-co-DBAEMA0.27) and PDBA were first dissolved in the deuterated buffers at a
concentration of 10 mg/mL, and 1,4-dioxane was used as an internal standard. To adjust the pH value,
0
.1 N NaOD deuterated solution was added and the Varian Mercury Plu600 MHz NMR spectrometer
1
was used to record the H-NMR spectra at different pH.
4
. Conclusions
A series of novel pH-responsive polymers based on tertiary amine functional groups were
developed by easy free radical copolymerization. The pK values of the polymers were precisely tuned
a
by adjusting the feed ratio between the two monomers, DMAEMA and DBAEMA. The phase transition
1
of polymers occurred in narrow pH ranges, which was demonstrated by transmittance and H-NMR
detection. Below pK
of the polymers in aqueous solution, whereas when pH was greater than pK
a
, the positive charges of the protonated amine groups maintained the solubility
, the hydrophobic butyl
a
groups on neutralized PDBAEMA segments rapidly induced the aggregation and precipitation of
the polymers. Compared to widely used PDMAEMA and PDEAEMA, the developed polymers
with PDBAEMA segments displayed much sharper pH transition ranges. The finely tunable
transition pH values mean the polymers are a promising platform for drug delivery and biomedicine
applications, where the encapsulated drugs at physiological pH would be triggered to release in
acidic microenvironments.
Supplementary Materials: The following are available online.
Author Contributions: H.F., P.L., and X.H. conceived and designed the experiments; P.L. and W.L. performed the
experiments; H.F. and X.H. analyzed the data; H.L. contributed reagents/materials/analysis tools; X.H. wrote
the paper.
Acknowledgments: This work was supported by the National Natural Science Foundation of China (Nos.
2
0474005, 51574267), the General Program of Science and Technology Development Project of Beijing Municipal
Education Commission (No. KM201310028007), the Fundamental Research Funds for the Central Universities
18CX05013), and Scientific Research Staring Foundation for the Returned Overseas Beijing Scholars (Grant No.
09125403700) and the Program for Changjiang Scholars and Innovative Research Team in University (IRT_14R58).
(
0
Conflicts of Interest: The authors declare no conflict of interest.
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