Thermo- and pH-Sensitivities of Thiosemicarbazone-Incorporated, Fluorescent and Amphiphilic Poly(N-isopropylacrylamide)

Thermo- and pH-Sensitivities of Thiosemicarbazone-

Incorporated, Fluorescent and Amphiphilic

Poly(N-isopropylacrylamide)

Cheng Li, Ling-Zhi Meng,* Xiao-Ju Lu, Zhao-Qiang Wu, Li-Fen Zhang, Yong-Bing He

Department of Chemistry, Wuhan University, Wuhan 430072, P. R. ChinaFax: (þ86) 27 68754067; E-mail: [email protected]

Received: May 28, 2005; Revised: July 8, 2005; Accepted: July 11, 2005; DOI: 10.1002/macp.200500222

Keywords: amphiphilic copolymer; fluorescent; pH-sensitive; poly(N-isopropylacrylamide); thermo-sensitive; thiosemicarbazone

Introduction

Amphiphilic copolymers tend to self-aggregate in selective

solvents to polymeric micelles that may provide a micro-

solvation media.[1,2] This interesting property has been

intensively investigated in the field of surfactants, nano-

structured materials, bioseparation, biomedicine, etc.[3–6]

Functionalizing of the micelles is an important factor in

achieving or enhancing these applications. Functional moie-

ties can be covalently linked to the water-soluble part of

the polymers or incorporated into the water-insoluble core

covalently or non-covalently. For drug carriers, an active

targeting, including chemical affinity targeting (molecules

such as sugar residues or antibodies) and physical affinity

targeting (pH-, thermo-, or magnetically responsive), might

mediate recognition and binding of the target thus allowing

the preparation of more efficient delivery systems.[7–9] In

addition, functional amphiphilic systems are potentially use-

ful for developing synthetic practical devices, for use in the

areas of material science, medicine, or in the chemical

laboratory.[10]

Thiourea or thiosemicarbazone functional moieties have

potential bioactivity. Their derivatives andmetal complexes

own pharmacological versatilities such as antibacterial,

antifungal, antitumoral, antiviral, anti-protozoa, or anticon-

vulsant.[11–13] Introducing a thiourea or thiosemicarbazone

Summary: Novel hydrophobic comonomer (p-methacryl-amido)acetophenone thiosemicarbazonewas synthesizedandpolymerized with N-isopropylacrylamide to get a series ofamphiphilic copolymers. The self-aggregation behavior andthermo-sensitive character of the (co)polymers were con-firmed by TEM observation, fluorescence spectra, and cloudpoint measurement. Fluorescence emission of copolymerwas significantly strengthened or switched off at an excitationwavelength of 320 nm upon the addition of acid or base,respectively. Thermo-sensitivity, pH-sensitivity, and pharma-cologically versatile thiosemicarbazone groups were inte-gratedintothesenovelfluorescentandamphiphiliccopolymers,which will develop the novel applications of amphiphiliccopolymer and environment-responsive materials.

Fluorescence intensity at 393 nm of polymer 2 in water at18 8C excited at 320 nm upon addition of acid and base.

Macromol. Chem. Phys. 2005, 206, 1870–1877 DOI: 10.1002/macp.200500222 � 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

1870 Full Paper

moiety as pendent group to a polymer backbone has been

attempted to get some uses by free radical polymerization or

macromolecular reactions.[14–17]

Poly(N-isopropylacrylamide) (PNIPAAm) is one of the

well-known thermo-responsive polymers. It exhibits a

lower critical solution temperature (LCST) in aqueous

media, below which the polymers are water-soluble and

above which they become significantly less water-soluble

or water-insoluble.[18] Due to the characteristic thermo-

sensitivity PNIPAAm has been widely investigated espe-

cially in biomedical field, for example, it was used as

cell culture membrane,[19] gene transfection vector,[20,21]

thermo-sensitive controlled release system,[22,23] antibody

purification,[24] etc. On the other hand, pH-sensitive poly-

mers have also been widely investigated, which usually

contain weak acid (–COOH) or weak base (–NH2, –NHR,

–NHR1R2) moieties, and the concept, kinds, sensitive

theories, and potential applications of pH-sensitive poly-

mers have been well reviewed.[25,26]

Here synchronously we introduced both the bioactive

thiosemicarbazone functional groups and fluorescent sig-

naling subunits into thermo-responsive PNIPAAm copoly-

mer. The resulting polymers are both thermo- and pH-

sensitive, and thefluorescent response can be easily realized

as the pH values changing in aqueous solution.

Experimental Part

Materials

N-Isopropylacrylamide (NIPAAm; Acros Organics; 99%) wasrecrystallized from mixtures of toluene and hexane (1/2 byvolume). Aminothiourea (Runjie, Shanghai; þ99%) andp-aminoacetophenone (Acros Organics; 99%) were used asreceived. N,N0-Azobisisobutyronitrile (AIBN; Shanghai) wasrecrystallized from methanol before use. Tetrahydrofuran(THF) was dried and distilled over Na before use. Methacryl-oyl chloride was prepared by refluxing a mixture of thionylchloride and methacrylic acid, followed by distillation.Adenosine 50-monophosphate disodium salt (Na2AMP �6H2O; ultra pure grade), adenosine 5

0-trisphosphate disodiumsalt (Na2H2ATP � 3H2O; ultra pure grade), and guanosine50-monophosphate disodium salt (Na2GMP; ultra pure grade)were purchased from Amresco Inc., USA. All the other saltsused are all analytical grade.

Synthesis of Thiosemicarbazone-ContainingHydrophobic Monomer II

3.37 g (37 mmol) aminothiourea was dissolved in 120 mL ofdeionized water. 1.5 mL of acetic acid was added and thesolution was then heated to about 75 8C. Under stirring 75 mLethanol solution of p-aminoacetophenone (5.0 g; 37 mmol)was added dropwise into the system. The reaction was allowedto stir for 15 h at a temperature of 85 8C. Most of ethanol wasremoved under reduced pressure. Then, cold deionized waterwas added to make the precipitation complete. The slight

yellow solid was filtered and washed with water, and vacuum-dried to give p-aminoacetophenone thiosemicarbazone (I)almost in quantitative yields.

1H NMR (300 MHz, CDCl3): d (ppm)¼ 2.24 (s, 3H, CH3),3.92 (s, 2H, PhNH2), 6.29 and 7.31 (each s, 1H� 2, S CNH2),6.65 and 7.54 (AA0BB0, 2H� 2, ArH), 8.68 (s, 1H, S CNH).

2.08 g (10 mmol) of I was dissolved in 50 mL of acetone.Later 0.89 mL (11 mmol) of pyridine was added and thesolution was then cooled by ice-salt bath. Under stirring 15mLof acetone solution of 1.06 mL (11 mmol) methacryloylchloride was added dropwise. The solution was stirred at roomtemperature for 5 h. The precipitation was removed by filtra-tion and the filtrate was concentrated under reduced pressure.Then a large amount of water was poured into it under stirringto precipitate the product. The collected product was repreci-pitated three times from acetone and placed under vacuum togive slight yellow powder (p-methacrylamido)acetophenonethiosemicarbazone (II), melting range: 201–204 8C.

IR (KBr): 3 420, 3 310, 3 272, 3 514, 1 658, 1 622, 1 593,1 533, 1 489, 1 449, 1 407, 1 285, 1 247, 1 088, 947, 840,542 cm�1.

1H NMR (300 MHz, CDCl3): d (ppm)¼ 1.98 (s, 3H,CH2 CCH3), 2.18 (s, 3H, N CCH3), 5.41 and 5.72 (each s,1H� 2, C CH2), 6.25 and 7.24 (each s, 1H� 2, S CNH2),7.50–7.62 (AA0BB0, 2H� 2, ArH), 7.52 (s, 1H, PhNH), 8.63(s, 1H, S CNH).

Elemental analysis forC13H16N4OS: Calcd. C 56.50, H 5.80,N 20.27, S 11.60; Found C 56.24, H 5.87, N 20.20, S 11.38.

FAB MS: m/z (RI)¼ 277 (MHþ, 45).

Copolymerization of NIPAAm and Hydrophobic Monomer II

In a typical synthesis, 1.13 g (10 mmol) of NIPAAm anddifferent amounts of hydrophobic monomer II were dissolvedinto 15 mL of dry THF, and initiator AIBN (at 0.9 mol-%relative to the monomers feed) was added. The solution wasthen purged with nitrogen under stirring. After heating to 65 8C,the reaction was run for 22 h under a nitrogen atmosphere.Most of THF was removed under reduced pressure and thesolution was poured into a large amount of cold diethyl ether toprecipitate the polymer. The collected polymer was reprecipi-tated three times by diethyl ether from THF and vacuum-driedat 40 8C for 24 h. A series of copolymer were synthesized bychanging the amount of hydrophobic monomer II.

IR (KBr): 3 435, 3 303, 2 973, 2 934, 2 876, 1 648, 1 541,1 458, 1 387, 1 367, 1 248, 1 173, 1 088, 840, 625 cm�1.

1H NMR (300MHz, CDCl3): d (ppm)¼ 7.70 (b, ArH), 6.56(b, S CNH2), 4.00 [b, NHCH(CH3)2)], 1.13 (b, CH3 ofNIPAAm and CCH3), 1.25–2.20 (b, main chain andN CCH3).

The general synthetic outline for polymerization is shown inScheme 1.

Characterization

1H NMR spectra were recorded in CDCl3 on a Varian MercuryVX-300 MHz spectrometer (USA). Fourier transform infrared(FT-IR) spectra were obtained using a Nicolet 670 FT-IR spectrometer (USA) and the samples were ground andpressed into KBr pellets for analysis. Elemental analysis was

Thermo- and pH-Sensitivities of Thiosemicarbazone-Incorporated, Fluorescent . . . 1871

Macromol. Chem. Phys. 2005, 206, 1870–1877 www.mcp-journal.de � 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

conducted on a Flash EA 1112 series elemental autoanalyzer(Italy). Mass spectra were obtained on a VJ-ZAB-3F massspectrometer (England) using the FAB technique. Meltingpoints were measured on a Reichert 7905 melting-pointapparatus (uncorrected).

The molecular weights and polydispersity indices of thepolymers were roughly estimated by gel permeation chroma-tography (GPC) analysis, using a Waters 2690-D liquid chro-matograph equipped with a Shodex K803 gel column and aninternalWaters 2410 refractive index detector. Chloroformwasused as eluent at a flow rate of 1.0 mL �min�1. Polystyrenestandard with a narrow distribution was used to generate acalibration curve.

LCST Determination of Thermo-Sensitive Polymers

The LCST values of thermo-sensitive (co)polymers wereestimated by cloud points (cp) measurements, which weredone visually by following the variation of the turbidity ofpolymeric aqueous solutions with temperature. Polymericaqueous solution (1.0 wt. %) in a sample tubewas immersed ina thermostated cell with a circulating water bath. The heatingratewas regulated around 0.1 8C �min�1 and the cpwas definedas the temperature at which the solution started to turn cloudy.The producibility of the determination was �0.5 8C.

LCST values were also determined by spectrophotometricdetection of the changes in transmittance (l¼ 500 nm) ofaqueous polymer solutions (4� 10�4 g �mL�1) heated at aconstant rate (0.2 8C �min�1). UV-vis absorption spectra wereacquired on a UV-240 UV-vis spectrophtometer (Shimadzu,Japan) equipped with a temperature-controlled circulatingwater bath. Values for the LCST of polymer solutions weredetermined as the temperature corresponding to the breakingpoint of optical transmittance.

Aggregation of Amphiphilic Copolymers in Water

The size and morphology of polymer particles in aqueoussolutions were determined by a JEM-100CXII transmissionelectron microscope (TEM; Hitachi, Japan). Dilute polymersolutions (4 mg �mL�1) were sonicated, then applied ontoformvar-membrane-coated copper grids and dried at 20 8C(below the LCST) to form a thin film for observation.

The self-aggregation of copolymer in water was fluoro-metrically investigated. The fluorescence spectra were deter-

mined as functions of polymer concentrations (3.3� 10�7 to1.7� 10�3 g �mL�1). From the results obtained the criticalconcentration of the polymer self-aggregation was estimated.The fluorometric measurements were recorded on a ShimadzuRF-5301PC spectrofluorophotometer (Shimadzu, Japan). Theslit settings were 5 or 10 nm and emission spectra weremonitored with an excitation wavelength of 320 nm (lex).

pH-Sensitivity of Copolymer in Water

The luminescence studies were carried out in water at 18 8C,which was below the LCSTof copolymer. Copolymer solutionof water (3.5mL; 5� 10�4 g �mL�1) was continuously titratedby hydrochloric acid (10�3 mol �L�1) or sodium hydroxide(10�3 mol �L�1) solution. From each run, relative fluorescenceintensity as a function of the volume of added Hþ or OH� wasrecorded. The pH values of the solutions were measured by apXSJ-216 ionanalyzer (REX1, Shanghai, China). A series ofcopolymer solutions inwater (3.5mL; 5� 10�4 g �mL�1)werealso added with increasing quantities of concentrated solutionsof salts. Being intermittently shaken, the mixed solutions wereincubated at 18 8C overnight for full interaction. Relativefluorescence intensity as a function of the concentration ofadded anion was recorded.

Results and Discussion

Polymer Synthesis and Characterization

Hydrophilic monomer NIPAAm was radically copolymer-

ized with hydrophobic comonomer II. Thiourea group

exists in a tautomeric equilibrium between the thione and

thiol forms and the latter is considered as a transfer agent in

radical polymerization. But careful study indicates that the

thiol form of thiourea only predominates in a strong acidic

media and its chain transfer reactivity is almost absent

above pH 3.5.[27] In this study the copolymerization was

carried out in a near neutral THF media; thus, the chain

transfer reactivity of thiourea residues was absent. Scheme

1 shows the general synthetic outline for polymerization.1H NMR, FT-IR, UV-vis absorption, and GPC were used

to characterize the polymers. The compositions and molec-

ular weights of the polymers are summarized in Table 1.

Scheme 1. Synthesis of copolymer.

Table 1. Results of polymerization and characterization.

Polymer m:n, feedratioa)

m:nb) Mnc) Mw=Mn

c)

g �mol�1

1 0:100 4 800 1.252 1:100 4 900 1.323 2:100 2:91 4 600 1.454 5:100 5:108 4 300 1.56

a) Feed molar ratio of monomer II to NIPAAm. The amount ofinitiator AIBN was 0.9 mol-% of total monomers.

b) Estimated from 1H NMR spectra.c) Determined by GPC.

1872 C. Li, L.-Z. Meng, X.-J. Lu, Z.-G. Wu, L.-F. Zhang, Y.-B. He


The 1H NMR spectrum of copolymer 3 is shown in

Figure 1. The characteristic peaks 4.8–6.5 ppm corre-

sponding to the vinyl groups of monomers disappeared

completely. Also peaks due to the –C6H4– groups (around

7.7 ppm) emerged. In FT-IR the number and positions of

substitutions on benzene ring can be related to the spectra.

In FT-IR spectra of polymer 2–4, the single and strong

absorbing band at 840 cm�1 originates from the motion of

bending of the C(ring)–H bond out of the plane of the ring,

and indicates the successful incorporation of hydrophobic

monomer II. Figure 2 gives out the UV-vis absorption of

polymer 1–4 and the monomer II. Each polymer displays

the n–p* transition at the band about 210 nm, due to the

C O bond originated from hydrophilic segment. Polymer

2–4 display p–p* transition at about 300 nm which

ascribed to the conjugation system of phenyl and Schiff

base from hydrophobic monomer II, with red shift from

295 nm of polymer 2 to 315 nm of polymer 4, whilePNIPAAm homopolymer 1 displays no p–p* transition.

With the increasing hydrophobicity of copolymers, the

water solubility became worse. Polymer 1–3 can easily

dissolve into water, and polymer 4 can dissolve into water,

while the polymer with the monomer molar ratio of 10:100

(not listed) cannot dissolve. The hydrophobic monomer IIhas a conjugated chromophore of the p–p conjugation of

phenyl and Schiff base, which made the copolymer show a

specific fluorescence in aqueous solutions with an excita-

tion maximum at about 320 nm and an emission maximum

at 393 nm. Moreover, hydrophilic moieties from NIPAAm

do not fluoresce excited at 320 nm in water and therefore do

not interfere. These properties meet the request of fluore-

scence detecting, and may be used to study copolymer self-

aggregation behavior and pH-sensitivity, which will be

discussed later.

Thermo-Sensitive Properties of (Co)polymers andEffect of Salts on LCST

The LCST value of PNIPAAm homopolyer (polymer I) isabout 32 8C in pure water, which matches to many refe-

rences. Little differences are found between the LCST

values obtained from visual observation and transmittance

determination and the latter are a little smaller (see Table 2,

polymers 2 and 4), which may be due to the relative

unreadiness of naked eyes. Significant decreases in LCST

were observed due to the introduction of hydrophobic

Figure 1. 1H NMR spectrum of copolymer 3 in CDCl3.

Figure 2. UV-vis absorption spectra of polymer 1–4 inH2O (10�4 g �mL�1). Inset: spectrum of monomer II in THF(10�5 mol �L�1).

Table 2. LSCT values of different (co)polymers.

Solution LCST values

8C

1 (0:100) 2 (1:100) 3 (2:100) 4 (5:100)

Water 32.0 29.5, 28.8a) 28.5 21.0, 20.2a)

NaClb) 32.0 28.0 27.0 18.0Phosphatec) 32.0 28.0 25.5 16.0

a) Values determined by optical transmittance.b) [NaCl]¼ 0.0322 mol �L�1.c) Phosphate consisted of KH2PO4 and Na2HPO4 whose pH (7.0)and ionic strength (I¼ 0.0322 mol �L�1) equal to those of NaClaqueous solution to draw comparisons.



groups (Table 2), increasing hydrophobes in polymer 1–4from 0 to 5.0 mol-%; the LCST’s decreased from 32 to

21 8C.The explanation for thismay be that with the increase

of hydrophobic/hydrophilic ratios, the hydrophobic effects

become stronger and the polymer chains tend to collapse;

thus, the polymers become less soluble in water and their

LCST’s decrease.[28–30]

On addition of salts, LCST’s of thermo-sensitive poly-

mers increased or decreased which was called ‘‘salting-in’’

or ‘‘salting-out’’ effect, respectively.[31,32] In this study,

NaCl and mixed phosphate buffer showed the salting-out

effect. The competition between intermolecular and intra-

molecular hydrogen bonding[33,34] and the hydrophobic-

hydrophobic interaction[35] can be used to explain these

from the viewpoint of mechanism. Homopolymer 1 shows

no decrease in LCST values with the addition of salts due to

thevery lowconcentration (0.0322mol �L�1) of salts, while

both NaCl and mixed phosphate exhibit stronger effects on

the thermo-sensitivity of amphiphilic polymer 2–4, and thesalts cause an increase of salting-out effect with the in-

creases of hydrophobicity (a decrease of 1–3 8C byNaCl or

1–5 8C by phosphate in LCST’s for polymer 2–4).

Self-Aggregation of Amphiphilic PNIPAAm

The self-aggregation behavior of the (co)polymers was

confirmed by TEM observation and fluorescence spectra.

Homopolymer PNIPAAm is fully hydrated with an ex-

tended chain conformation in aqueous solution, and

coil-globule transition of single chain emerges when the

temperature T> y (y is the temperature where the second

virial coefficient becomes zero)[36]. For PNIPAAm y is

about 30.59–30.71 8C independent of the molecular

weight.[36] TEM photos (not given here) of polymer 1 and

3were all obtained at the temperature far below the LCST’s.

PNIPAAm homopolymer 1 is random coil, indicating an

extended chain conformation in the good solvent region,

while the amphiphilic polymer 3 shows a globule-like

morphology, indicating self-aggregation and micelle

formation.

The synthesized copolymers show a specific fluores-

cence in aqueous solution with an excitation maximum at

about 320 nm and an emissionmaximum at 393 nm, so they

can be considered as polymers with intrinsic fluorophores.

It is known that the fluorescence behavior of this intrinsic

probe would reflect the dynamics of the polymer back-

bone.[37] Figure 3 gives out the fluorescence intensity de-

pendence of polymer 3 on polymer concentration. The

fluorescence intensity increases as polymer concentration

reaches a maximum and then undergoes a slight decrease.

This maximum (about 7.0� 10�4 g �mL�1) could be the

critical micelle concentration (CMC), where the amphi-

philic polymer coils associate to form large aggregates.

Above it, the concentration of fluorophore will be high

enough in the interchain hydrophobic domains to cause

self-quenching of fluorescence.[38,39] If there are only one

or two fluorophores per polymer, the fluorescence increases

as the polymer wraps up the fluorophore in an increasingly

viscous and oily environment by reducing the energy lost to

surrounding water molecules. However, if several fluoro-

phores are pulled together, the energy may be transferred

between fluorophores, either by re-absorption or by non-

radiative energy transfer,[40] while below the CMC, fluores-

cence intensity does not increase linearly as the concen-

tration increases. This indicates that some intrachain

hydrophobic domains will be still formed at concentration

below the CMC due to intramolecular hydrophobic

interaction.

pH-Sensitivity of Copolymer in Water

Thepolymer synthesized in this studycontains theNIPAAm

moieties as its hydrophilic segments and the thiosemicar-

bazone moieties as its hydrophobic segments. Thus, the

polymer is thermo-sensitive due to NIPAAm units as dis-

cussed before. In addition, the weak basic thiourea residues

in the hydrophobic segments could influence the hydro-

phobicity of the thiosemicarbazone moiety to make the

polymer also pH-sensitive.

Figure 4 shows the fluorescence intensity of polymer 2upon the addition of acid or base. With the decrease of the

pH of the polymer solution (volume increase of acid was

added), the fluorescence intensity increased and reached a

maximum as about 80 mL acid added (pH� 6.5), while the

pH increase (base addition) made the fluorescence intensity

decrease and reach a minimum as about 180 mL base was

added (pH� 8.5). Thus, the thiosemicarbazone-incorpo-

rated polymer is pH-sensitive between pH¼ 6.5 and 8.5.

This is a comparatively narrow pH-sensitivity, but this pH

Figure 3. Fluorescence emission spectra (slit settingswere5nm)of polymer 3 with different concentrations (C) (10�6 to 1.71�10�3 g �mL�1) at 20 8C excited at 320 nm. Inset: fluorescenceintensity (at 393 nm) dependence on polymer concentration C.



range accords with physiological condition of human body

and may have potential medicinal application.

The factors contribute to the triggering of this pH-

sensitivity may be the hydrophilic segment (due to the

variation of the amide-water hydrogen bonding), or the

hydrophobic segment (due to the protonation/de-protona-

tion of the thiourea group), or both of them. First, the addi-

tion of acid (pH-decrease) significantly increased the

amide-water hydrogen bonding and hence the solvation of

polymer chains, which in turn would increase the water

solubility of the polymer. Second, pH-decreasewouldmake

the thiourea residue more protonated; the tautomeric equi-

libriumof it in acidic aqueous solution is shown in Scheme2

where K is the tautomeric equilibrium constant.[27] As a

result, the electrostatic repulsion between adjacent cations

increased and the polymer chain expanded. Both of these

two forces expanded the polymer chain and the covalently

attached chromophores were separated, leading to decreas-

ed excited-state energy transfer and increased fluorescence

intensity. Although such expansion and separation was

limited, the fluorescence intensity reached a maximum and

remained constant. On the contrary, the addition of base

(pH-increase) decreased the amide-water hydrogen bond-

ing and enhanced the hydrophobic effect. Thus, the polymer

chain contracted more and more and brought the chromo-

phores closer, hence increasing the excited-state energy

transfer and decreasing fluorescence intensity till the

polymer is insoluble in water.

This pH-sensitivity can also present upon the addition of

different salts. Eleven kinds of sodium salts were, respec-

tively, added into the aqueous solution of polymer 3, viz.,Cl�, Br�, HPO4

2�, H2PO4�, CO3

2�, HCO3�, CH3COO

�,

p-C6H4(COOH)COO�, GMP2�, AMP2�, and H2ATP

2�,

and all the tests run at the temperature far below the LCST’s

whether the salts were added or not. Results showed that on

the addition of five kinds of anionic salts, viz. HPO42�,

CO32�, HCO3

�, GMP2�, and AMP2� the fluorescence

intensity decreased by various degrees. The typical fluo-

rescence-quenching curve of CO32� is shown in Figure 5,

and the relative fluorescence intensity dependence on salts

concentration in water is given in Figure 6. The other six

kinds of salts of anions, Cl�, Br�, H2PO4�, CH3COO

�,

p-C6H4(COOH)COO�, andH2ATP

2�, have no influence on

the fluorescence intensity of polymer. As an example no

fluorescence spectra difference was shown between aque-

ous solution and 0.0322mol �L�1 NaCl solution of polymer

3. Some reasons may contribute to the quenching behavior

and the dissociation constants (pK) of the added salts may

be a primary one. Except for Cl� and Br� the anions of the

added salts are all weak acidic anions and they will disso-

ciate in water as a conjugate base. The base dissociation

constants (pKb) of CO32�, GMP2�, HCO3

�, and AMP2� are

3.75, 7.49, 7.63, and 7.94, respectively.[41] So, the bases

become stronger in the order of CO32�>GMP2�>

HCO3�>AMP2�. The order accords with the order in

which the salts quenched the fluorescence intensity. The

Figure 4. Fluorescence intensity at 393 nmof 3.5mLof polymer2 (5.0� 10�4 g �mL�1) in water at 18 8C excited at 320 nmupon addition of acid (HCl, 10�3 mol �L�1) and base (NaOH,10�3 mol �L�1) (with slit settings of 10 nm). Numbers beside thedata points are the relevant pH values.

Scheme 2. Tautomeric equilibrium of the thiourea residue.

Figure 5. Emission spectra of of polymer 3 (5.0� 10�4 g �mL�1) inwater upon addition ofCO3

2�. Spectrawere all recoded at18 8C and excited at 320 nm with slit settings of 5 nm.



pKb values of CH3COO�, p-C6H4(COOH)COO

�, H2PO4�,

and H2ATP2� are 9.25, 11.02, 11.88, and 12.3, respectively.

They are very weak bases and their water solutions are a

near neutral pH media. Thus, they did not influence the

fluorescence intensity of polymer within the experimental

range. It should be noted that 0.0322 mol �L�1 Cl�

decreased the LCST’s of polymer 2–4 [see part Thermo-

Sensitive Properties of (Co)Polymers and Effect of Salts on

LCST] while in such concentration fluorescence intensity of

polymer 3 remained unchanged (figure not given). This

reflected that Cl� induced a sharp phase transition at critical

concentration, which is not a typical salting-out behavior

(gradually phase transition with increasing salt concen-

tration).[42] Below the critical Cl� concentration and at

experimental temperature the polymer coils did not gradu-

ally collapse and kept the fluorescence intensity constant.

The other anions that did not influence the fluorescence

intensity in this study may also own this behavior. It is also

of interest to note that in the experiment anion HPO42�,

whose pKb is 6.8 between CO32� and GMP2�, showed

a weaker fluorescence quenching ability than it should

have. The rationales behind our present observations of

the salt effect on the fluorescence behavior of thermo-

sensitive copolymer are more complex and deserve further

studies.

Conclusion

Aseries of amphiphilic, fluorescent, and thermo-responsive

copolymers were prepared and characterized. The self-

aggregation behavior and thermo-responsive characters of

the copolymers were confirmed by TEM observation, fluo-

rescence spectra, and cp measurement. The fluorescence

intensity of amphiphilic polymer 3 increases with concen-

tration and reaches a CMC of about 7.0� 10�4 g �mL�1.

The polymers are thermo-sensitive, andwith the increase of

hydrophobic/hydrophilic ratios, the polymers show lower

LCST’s and are more affected by addition of salts. The

copolymers are also pH-sensitive as fluorescence emission

of polymer 3was significantly strengthened or switched offupon the addition of acid or base, respectively. And it was

also observed as some anions were added into polymer

aqueous solutions. That is a comparatively narrow pH-

sensitivity (pH¼ 6.5–8.5), but the pH range accords with

the physiological condition and may have potential medi-

cinal application. Thermo-sensitivity, pH-sensitivity, and

pharmacologically versatile thiosemicarbazone groups are

integrated into these novel fluorescent and amphiphilic

copolymers, which will develop the novel applications

of amphiphilic copolymer and environment-responsive

materials.

Acknowledgements: The authors are grateful to Ms. Gui-RongZhong and Ms. Xi Yang, Department of Chemistry, WuhanUniversity, for their help in TEM observation and fluorescencespectra determination, respectively. They thank the NationalNatural Science Foundation for financial support (Grant No.20474044).

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2�, (~) HCO3�, (*) CO3

2�, (!) AMP2�, (^) GMP2�.



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