Use of Core-Cross-Linked Polymeric Micelles Induced by the

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Use of Core-crosslinked Polymeric Micelles Induced by the Selective Detection of Cu(II) Ions for the Sustained Release of a Model Drug Jae Min Bak, and Hyung-il Lee ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b02432 • Publication Date (Web): 27 Mar 2019 Downloaded from http://pubs.acs.org on April 2, 2019

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ACS Applied Materials & Interfaces

Use of Core-crosslinked Polymeric Micelles Induced by the Selective Detection of Cu(II) Ions for the Sustained Release of a Model Drug Jae Min Bak and Hyung-il Lee*

Department of Chemistry, University of Ulsan, Ulsan 680-749, Republic of Korea

*E-mail: [email protected]

Keywords. Cu(II) ions, Polymeric micelle, Colorimetric sensor, Sustained drug release, Thiosemicarbazone

ACS Paragon Plus Environment

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Abstract A well-defined amphiphilic phenylthiosemicarbazone-based block copolymer was successfully synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization,

followed

by

post-polymerization

modification.

Poly(N,N-

dimethylacrylamide) (pDMA) was synthesized via RAFT polymerization of N,Ndimethylacrylamide (DMA). The resulting pDMA macro chain transfer agent was further extended using 3-vinylbenzaldehyde (VBA) to yield the poly[(N,N-dimethylacrylamide)b-(3-vinylbenzaldehyde)] [p(DMA-b-VBA)] block copolymer. The aldehyde groups of p(DMA-b-VBA) were then made to react with 4-phenylthiosemicarbazide to yield the target

block

copolymer

poly{N,N-dimethylacrylamide-b-[N-phenyl-2-(3-

vinylbenzylidene)hydrazine carbothioamide]} [p(DMA-b-PVHC)]. p(DMA-b-PVHC) self-assembled in aqueous solution to yield polymeric micelles that comprise a pDMA block that forms a hydrophilic shell and a pPVHC block that forms a hydrophobic core. p(DMA-b-PVHC) micelles can detect Cu(II) ions which can be determined by a color change from colorless to yellow induced by the formation of coordination complexes between Cu(II) ions and the phenylthiosemicarbazone units of p(DMA-b-PVHC). As Cu(II) ions slowly penetrated the core of p(DMA-b-PVHC) micelles, these cores crosslinked with each other, which in turn resulted in the micelle particles swelling in water. Upon the addition of Cu(II) ions to a solution of p(DMA-b-PVHC) micelles encapsulating the hydrophobic model drug coumarin 102, this drug was released from the micelles in a sustained manner due to the gradual swelling of the crosslinked micelle cores caused by the slow penetration of Cu(II) ions.


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Introduction Drug delivery systems based on polymers that allow for controllable and sustained drug release have attracted significant attention, since they can potentially be used to treat numerous diseases, including cancer and diabetes.1-6 Due to their ease of functionalization, low cost, and possibility of mass production, polymers are promising starting points in the development of drug delivery systems.7-8 Sustained drug delivery has recently become a key objective in the development of pharmaceutical products.9-14 Technologies that render possible the sustained release of drugs enhance the performance of compounds with pharmaceutical properties by regulating the temporal drug profile to maximize the compound’s therapeutic benefits.15 In fact, when such systems are utilized, patients benefit from less frequent medication injections, enhanced therapeutic efficacy brought about by an extension of the duration of the activity, and a reduction in the prevalence and intensity of side effects. Several types of polymeric materials have been exploited to effect sustained release, including transdermal patches, hydrogels, starshaped polymers, and dendrimers.16-19 Sustained drug delivery systems can also be based on polymeric micelles constructed by amphiphilic block copolymers.20-23 The main problems associated with the use of block copolymer micelles are these systems’ spontaneous dissociation at concentrations below the critical micelle concentration (CMC) and the premature leakage of drugs from micelles as the drug delivery systems travel in the vascular system. To solve these problems, various methods for the cross-linking of polymeric micelles have been proposed.24-25 In this context, crosslinks can be formed in the core or in the shell of the micelles.


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Recently, the selective detection of transition metal ions has become increasingly important. In particular, sensing Cu(II) ions has attracted special attention because these ions have both positive and negative effects on human health and the environment.26-27 While numerous methods have been developed to detect Cu(II) ions effectively, colorimetric detection via the formation of complexes between ligands and Cu(II) ions has been proposed by many researchers.28-35 We recently reported a method for the selective colorimetric sensing of Cu(II) ions in aqueous media based on the use of polymeric probes with phenylthiosemicarbazone units as ligands.36 In recent years, efforts have been made to merge advances in sensor technology with those in drug delivery systems, because the continuous monitoring of specific target species is often needed to achieve efficient disease treatment. The integration of sensing and drug delivery systems is an efficient approach to control drug release. For example, numerous studies have demonstrated the presence of high copper ion levels in tumorbearing mice as well as in the tumors and blood serum of patients with cancer.37-39 In fact, high serum copper levels have been correlated with the presence of a plethora of cancers.40-43 Whenever high serum copper levels are detected, therapeutic dosing from drug carriers can be controlled. Herein, we report the synthesis of poly{[N,N-dimethylacrylamide]-b-[N-phenyl-2-(3vinylbenzylidene)hydrazinecarbothioamide]} [p(DMA-b-PVHC)] block copolymer, its self-assembly to form polymeric micelles in water, and the sustained release of the hydrophobic model drug coumarin 102 encapsulated in the crosslinked core of the micelles thus generated, which is driven by the turn-on colorimetric detection of Cu(II) ions. In this study, therefore, p(DMA-b-PVHC) block copolymer was used as a


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chemosensor for the colorimetric detection of Cu(II) ions as well as a delivery vehicle for the sustained release of coumarin 102.

Experimental Materials. 2-(Dodecylthiocarbonothioylthio)-2-methylpropionic acid (DMP, 98%), 2,2′azobisisobutyronitrile

(AIBN,

98%),

N,N-dimethylformamide

(DMF,

99.8%),

tetrahydrofuran (THF, 99.9%), 1,3,5-trioxane (99%), N-(2-Hydroxyethyl)piperazine-N′(2-ethanesulfonic acid) (HEPES), and all the metal salts with the highest purity available were purchased from Aldrich and used as received. 4-Phenylthiosemicarbazide (98.0%) was purchased from TCI (Tokyo Chemical Industry) and used as received. 3Vinylbenzaldehye (VBA, Aldrich, 97%) and N,N-dimethylacrylamide (DMA, TCI, 99.0%) were passed through a column of basic alumina before their use in the polymerization reaction. Instrumentation. 1H nuclear magnetic resonance (NMR, Bruker Avance 300 MHz NMR) spectroscopy experiments were performed in CDCl3. Gel permeation chromatography (GPC, Agilent technologies 1200 series) was conducted using a polystyrene standard with DMF as the eluent at 30 °C and a flowrate of 1.00 mL/min. The UV-Vis spectra were recorded using a SINCO Mega Array PDA UV-Vis spectrophotometer. Hydrodynamic size distributions were determined by dynamic light scattering (DLS, Nano ZS, Malvern, UK). Fluorescence emission spectra were recorded with a HORIBA FluoroMax-4Pmspectrophotometers. Atomic force microscopy (AFM) images were obtained using a NX10 AFM device (Park systems, Suwon, Korea).


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Synthesis of poly(N,N-dimethylacrylamide) (pDMA). DMA (9.60 ml, 97.04 mmol), DMP (176.9 mg, 0.485 mmol), AIBN (1.99 mg, 0.012 mmol), 1,3,5-trioxane (43.6 mg, 0.485 mmol, internal standard), and DMF (10.0 mL) were added to a 25 mL Schlenk flask containing a magnetic stir bar, and the flask sealed immediately after all compounds were added to it. The solution thus obtained was purged with argon for 20 min, and the reaction flask was placed in an oil bath preheated to 70 °C. After 1 h, the polymerization was quenched by removing the reaction flask from the oil bath and exposing the solution to air. The solution was precipitated into cold ether. The polymer was then re-dissolved in THF and reprecipitated into the cold ether and dried under vacuum at room temperature for 24 h. Mn = 21 500 g/mol, Mw/Mn = 1.06. 1H NMR (300 MHz, CDCl3, δ in ppm): 2.85 (6H, s, -N(CH3)2); 2.00-1.00 (3H, m, -CH2CH-); 0.85 (3H, t, -CH3) Synthesis of poly[(N,N-dimethylacrylamide)-b-(3-vinylbenzaldehyde)] [p(DMA-bVBA)]. p(DMA-b-VBA) block copolymer was synthesized by reversible addition– fragmentation chain transfer (RAFT) polymerization using pDMA as a macro chain transfer agent (macroCTA). VBA (0.164 ml, 1.29 mmol), pDMA (0.31 g, 0.026 mmol), AIBN (1.06 mg, 0.00645 mmol), 1,3,5-trioxane (23.2 mg, 0.26 mmol, internal standard), and DMF (5 mL) were added to a 10 mL Schlenk flask containing a magnetic stir bar, and the flask sealed immediately after all compounds were added to it. The solution was then purged with argon for 20 min, and the reaction flask was placed in an oil bath preheated to 70 °C. After 3h, the polymerization was quenched by removing the flask from the oil bath and exposing the solution to air. The solution was precipitated into cold ether. The polymer was the re-dissolved in THF and reprecipitated into the cold ether and dried under vacuum at room temperature for 24 h. Mn = 24 400 g/mol, Mw/Mn = 1.07. 1H


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NMR (300 MHz, CDCl3, δ in ppm): 9.95 (1H, s, -CHO); 7.80-6.4 (4H, s, ArH); 2.85 (6H, s, 2.85 (6H, s, -N(CH3)2); 2.00-1.00 (3H, m, -CH2CH-); 0.85 (3H, t, -CH3) Synthesis

of

poly{[N,N-dimethylacrylamide]-b-[N-phenyl-2-(3-

vinylbenzylidene)hydrazinecarbothioamide]}

[p(DMA-b-PVHC)].

A

post-

polymerization modification reaction was conducted as previously reported.36 Briefly, p(DMA-b-VBA)

(0.179

g,

0.135

mmol

per

VBA

repeating

unit)

and

4-

phenylthiosemicarbazide (45.2 mg, 0.270 mmol) were added to a round-bottom flask and dissolved in THF (50 mL); the resulting solution was then heated at 80 °C overnight. The solution was concentrated and precipitated in diethyl ether. The precipitation process was repeated two times and the precipitate thus obtained was dried in a vacuum oven at 30 °C for 24h. Mn = 24 500 g/mol, Mw/Mn = 1.08. 1H NMR (300 MHz, CDCl3, δ in ppm): 9.60 (1H, s, −NH); 9.0 (1H, s, −NH); 7.80 (1H, s, −CHN−); 7.80−6.80 (9H, m, ArH).


7


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Results and Discussion

HOOC

S

S

C12H25

HOOC

S

S

O

n

O

AIBN, DMF, 70oC

N

S

H C12H25

O

S

AIBN, DMF, 60oC

N

pDMA

HOOC

b n

O

S m

S

HOOC

S C12H25

S

N

N H

N H

b n

NH2

O

S m

S

C12H25

S

N

THF, 80oC, reflux

N

O HN

NH S

p(DMA-b-VBA) p(DMA-b-PVHC)

Scheme 1. Synthesis of p(DMA-b-VBA) via RAFT homopolymerization of DMA and subsequent RAFT block copolymerization of 3-VBA, followed by post-polymerization modification of p(DMA-b-VBA) to yield the amphiphilic block copolymer p(DMA-bPVHC), which contains thiocarbazone moieties. 3-VBA: 3-vinylbenzaldehye; AIBN: 2,2′-azobisisobutyronitrile; DMA: dimethylacrylamide; DMF: N,N-dimethylformamide; PDMA: poly(N,N-dimethylacrylamide; p(DMA-b-VBA): poly[(N,N-dimethylacrylamide)b-(3-vinylbenzaldehyde)];

p(DMA-b-PVHC):

poly{[N,N-dimethylacrylamide]-b-[N-

phenyl-2-(3-vinylbenzylidene)hydrazinecarbothioamide]}; RAFT: reversible addition– fragmentation chain transfer.

The synthetic strategy adopted in this study is depicted in Scheme 1. RAFT polymerization of DMA, with DMP acting as a chain transfer agent and AIBN acting as


8

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an initiator, led to the production of pDMA with a well-controlled molecular weight and a low level of polydispersity. Polymerization was carried out at 70 °C in conditions whereby [DMA]:[DMP]:[AIBN] = 200:1:0.025 and in the presence of 1,3,5-trioxane as an internal standard. Polymerization was quenched when DMA monomer conversion reached 60%, which occurred after about 1 h. Values for the apparent molecular weight (Mn,

app

= 21,500) and molecular weight distribution (Mw/Mn = 1.06) of the resulting

polymer pDMA were determined by GPC (Figure 1). Notably, the apparent molecular weight obtained by GPC was higher than the theoretical molecular weight (Mn, 12,200, DPn,

theory

theory

=

= 120) calculated on the basis of DMA monomer conversion. The

experimental molecular weight of pDMA can also be determined by

1H

NMR

spectroscopy. Mn, NMR was determined based on the integration of the resonance signals at 2.85 ppm, which is due to the dimethyl groups of DMA repeating units, and at 0.85 ppm, which due to the DMP end groups (-CH3). Notably, the value of Mn, NMR (11,800 g/mol) was in relatively good agreement with that value of Mn, theory (Figure 2a).


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p(DMA)120 p(DMA120-b -VBA10) p(DMA120-b -PVHC10)

4

10

5

Molar Mass

10

Figure 1. Overlaid GPC traces of pDMA, p(DMA-b-VBA), and p(DMA-b-PVHC).


10

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Table 1. Yields of the syntheses of pDMA, p(DMA-b-VBA), and p(DMA-b-PVHC) performed via reversible addition-fragmentation chain transfer polymerization, followed by post-polymerization modification, and characteristics of the polymers thus obtained Polymer

Conva (%)

Mn, theoryb

Mn, NMRc

Mn, appd

PDId

pDMA120 p(DMA120-b-VBA10) p(DMA120-b-PVHC10)

60 20 -

11 800 13 100 -

12 200 13 500 15 000

21 500 24 400 24 500

1.06 1.07 1.08

Monomer conversion determined by 1H NMR spectroscopy analysis. b Theoretical molecular weight determined on the basis of monomer conversions. c Experimental molecular weight calculated from 1H NMR spectroscopy data. d Apparent number-average molecular weight and PDI determined by DMF GPC with polystyrene calibration. a

HOOC

S

b

S

m

n

O

S

N

l N

(c)

p(DMA-b -PVHC) p(DMA-b -VBA) pDMA

CDCl 3

C 12 H 25

NH

k HN

j

11 10 HOOC

O

(b)

S

b n

N

h g

m

i

S

(a) 11

bc O

N

10

8

7

6

C 12 H25

e

f

O

f+g+h+i S

n

9

S

e HOOC

l

k j

S

S

(C 11H 22 )

S

a

CH 3

d

b

a

9

8

7

6

ppm

5

4

3

2

c

d

1

0

Figure 2. 1H NMR spectra of (a) pDMA, (b) p(DMA-b-VBA), and (c) p(DMA-bPVHC).

RAFT polymerization of VBA was initiated using the well-defined pDMA as macroCTA. The polymerization was performed in DMF, and the [VBA]:[pDMA


11


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mcaroCTA]:[AIBN] ratio used was 50:1:0.25. After 3 h, the extent of VBA conversion was 15%, resulting in the formation of the p(DMA-b-VBA) block copolymer with Mn = 13,100 and Mw/Mn = 1.07. The increase in molecular weight of p(DMA-b-VBA) with respect to pDMA is demonstrated by the slight shift of the GPC traces toward higher molecular weight. Notably, following p(DMA-b-VBA) formation, the polymer’s molecular weight distribution remained relatively narrow, confirming the idea that this synthetic approach enabled researchers to exercise an effective control over the block copolymerization (Figure 1 and Table 1). Besides relying of GPC data, the experimental molecular weight of p(DMA-b-VBA) was also calculated using 1H NMR spectroscopy data, by determining the value of the integral of the 2.85 ppm resonance peak due to the DMA repeat unit (-N(CH3)2) and of the resonance peak at 9.95 ppm due to the VBA repeat unit (-CHO). In this case, Mn, NMR = 13,500, where the value of DPNMR of DMA and VBA is 120 and 10, respectively (Figure 2b). Having obtained the p(DMA-b-VBA) block copolymer, a post-polymerization modification reaction was conducted to yield p(DMA-b-PVHC), a species comprising thiocarbazone moieties. 4-Phenylsemithiocarbazide was successfully coupled to p(DMAb-VBA) with high efficiency. To ensure full conversion, a two-fold excess of 4phenylsemithiocarbazide with respect to the number of aldehyde groups present in the VBA block in p(DMA-b-VBA) was used, and the reaction was allowed to proceed at 80 °C for 12 h. 1H NMR spectroscopic analysis confirmed the successful transformation of p(DMA-b-VBA) into p(DMA-b-PVHC). In fact, following this procedure, new peaks (i, j, k) appeared in the 7.8–10.0 ppm range and an aldehyde peak (e) that was present in the spectrum of p(DMA-b-VBA) disappeared completely. The reaction was almost


12

Page 13 of 29

quantitative, with an essentially complete conversion of the aldehyde units being observed. The GPC traces indicated that no significant change in the apparent molecular weight of the polymer had occurred as a consequence of the coupling reaction (Figure 1).

a)

b)

1.5

Cu(II) Cu(II)

250

c)

No metal cations

1.0

Cu(II) 100 M Zn(II) 100 M Ni(II) 100 M Pb(II) 100 M

0.5

500

0.0

Cr(II) 100 M Co(II) 100 M Fe(II) 100 M Cd(II) 100 M Mn(II) 100 M

250 300 350 400 450 500 550 600 Wavelength (nm)

[Cu(II) 100 M]

1.5

0h 10 min 1h 3h 5h 9h 24 h

1.0

0.5

0.0

300 350 400 450 Wavelength (nm)

Absorbance

Absorbance

0.5

0.0

1.5

[Cu(II)] 0 M 33 M 66 M 100 M

1.0

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


250

300 350 400 450 Wavelength (nm)

500

Figure 3. a) Room-temperature UV-Vis absorption spectra of 0.1 mg/mL p(DMA-bPVHC) aqueous micellar solutions (66 μM of the phenylthiosemicarbazone units) recorded right after the addition of Cu(II) ion solutions to reach various final metal ion concentrations. b) Cu(II)-ion selectivity of p(DMA-b-PVHC) micelles in the presence of various other metal ions.(inset: from left to the right, no metal, Cu(II), Zn(II), Ni(II), Pb(II), Cr(II), Co(II), Fe(II), Cd(II) and Mn(II)) c) Time-dependent change in UV-Vis absorption spectra of a 0.1 mg/mL p(DMA-b-PVHC) micellar solution to which had been added Cu(II) ions to a final concentration of 100 μM. d) Schematic representation of the


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time-dependent detection of Cu(II) ions resulting from the metal ions’ slow penetration into a hydrophobic PVHC core.

The p(DMA-b-PVHC) block copolymer is amphiphilic and, in aqueous solution, it can self-assemble to form micelles. The CMC value for this copolymer was determined as previously reported using a fluorescence technique and pyrene as a probe.44 The value thus obtained was 6.6  10−3 mg/mL (Figure S1). The Cu(II)-ion-sensing studies using self-assembled p(DMA-b-PVHC) micelles were conducted in aqueous solution. In particular, an aqueous 0.1 mg/mL micellar solution of p(DMA-b-PVHC) was prepared that was characterized by a 66 μM concentration of phenylthiosemicarbazone units. To ensure the successful formation of micelles, p(DMAb-PVHC) was micellized at a concentration that was much higher than the CMC. Subsequently, changes in the features of the UV-Vis absorption spectra of p(DMA-bPVHC) micelles resulting from the gradual addition to the micellar solution of Cu(II) ions to a final 33–100 μM metal ion concentration range were monitored (Figure 3a). The gradual decrease in intensity of the absorption maximum at 318 nm and the gradual increase in absorbance at 390 nm where is the onset of the greenish-yellow color, coupled with the overall broadening of the absorption band were observed upon the addition of the Cu(II) ion solution. Furthermore, the originally colorless solution became yellow due to the formation of coordination complexes between Cu(II) ions and the phenylthiosemicarbazone units of p(DMA-b-PVHC) micelles.36, 45 As previously reported, p(DMA-b-PVHC) exhibited good selectivity toward Cu(II) ions in the presence of several alkali and transition metal cations (Figure 2b). Interestingly, the Cu(II) ion detection


14

Page 15 of 29

process is time-dependent. Changes in the UV-Vis absorption spectra of p(DMA-bPVHC) micelles as a function of time, following addition of Cu(II) ions to a final concentration of 100 μM, were monitored (Figure 3c and see also Figure S2a and S2b for different final concentrations of Cu(II) ions). It was found that the time required for the complete detection of Cu(II) ions with p(DMA-b-PVHC) micelles was about 24 h. The detection time profile of p(DMA-b-PVHC) micelles toward Cu(II) ions present at different concentrations was also plotted (Figure S2c). The hydrophilic Cu(II) ions have limited access to the inner core of the micelles, where the pPVHC blocks with phenylthiosemicarbazone units are closely packed. As time progressed, however, Cu(II) ions slowly penetrated into the hydrophobic pPVHC core of the micelles, resulting in the time-dependent formation of coordination complexes (Figure 3d).

0h 6h 18 h 24 h

a)30 25

Volume (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


20 15 10 5 0

10

100

Hydrodynamic Diameter (nm)

Figure 4. a) Time-dependent increase in apparent micellar hydrodynamic diameter of a 0.01 mg/mL p(DMA-b-PVHC) micellar solution following the addition to the said solution of Cu(II) ions to a final metal cation concentration of 100 μM. b) Mechanism of formation of core-crosslinked p(DMA-b-PVHC) micelles with ionic cores via the intermolecular

tetradentate

coordination

complexation

by

p(DMA-b-PVHC)’s

phenylthiosemicarbazone units with Cu(II) ions.


15


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DLS experiments were conducted to monitor the formation of p(DMA-b-PVHC) micelles as well as the change in micelle size resulting from the formation of coordination complexes between these micelles and the Cu(II) ions. In these experiments, Cu(II) ions to a final concentration of 100 μM were added to an aqueous 0.01 mg/mL micellar solution of p(DMA-b-PVHC), and the evolution of the micellar size distribution was measured as time progressed (Figure 4a). The average hydrodynamic diameter of the original micelles was 40 nm, indicating that p(DMA-b-PVHC) self-assembled to form micellar aggregates. After addition to the micellar solution of Cu(II) ions to a final concentration of 100 μM, the average hydrodynamic diameter of the micelles increased gradually over time, reaching a value of 75 nm after 24 h. It has been reported that Cu(II) ions tend to form bidentate coordination complexes with phenylthiosemicarbazone.28, 45 However, the formation of tetradentate coordination complexes is facilitated in the case of p(DMA-b-PVHC) micelles, because phenylthiosemicarbazone units are densely packed in the inner core of micelles. The slow penetration of Cu(II) ions into the core of the micelles led to the intermolecular tetradentate coordination complexation of the metal ions by phenylthiosemicarbazone units. In turn, this complexation induced the formation of polymer micelles with crosslinked ionic cores (Figure 4b). As a result, the average hydrodynamic micellar diameter gradually increased over time as a consequence of the swelling of crosslinked ionic cores in water.


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Figure 5. Representative AFM phase images of a 0.01 mg/mL p(DMA-b-PVHC) micellar solution spin-coated on mica before (a) and after (b) the addition to it of Cu(II) ions to a final concentration of 10 μM.

The Cu(II) detection-induced formation of core-crosslinked p(DMA-b-PVHC) micelles was confirmed by evidence from AFM measurements. In particular, these microscopy experiments were performed by preparing samples from an aqueous solution of p(DMAb-PVHC) micelles (0.01 mg/mL) that was deposited directly on a mica substrate by way spin-coating conducted after micellization. The uniform, well-dispersed individual spherical micelles with an average diameter of 35 nm that were obtained implementing the described procedure were visualized by AFM (Figure 5a). Cu(II) ions to a final concentration of 10 μM were then added to the p(DMA-b-PVHC) micelle solution (0.01 mg/mL), and 24 h later the resulting solution was spin-coated onto a mica substrate for AFM analysis. Although evidence indicates that after implementation of the process just described the characteristic micellar morphology was retained, the average diameter of the globular particles increased to 83 nm (Figure 5b). In fact, as a consequence of Cu(II)


17


ion coordination, the cores of p(DMA-b-PVHC) micelles were crosslinked to each other, leading to the swelling of the micelle particles.

6

1.0x10

5

5.0x10

0.0 400

0 1min 1h 1day 2day 3day 7day 10day 15day 21day 1 month

500 600 700 Wavelength (nm)

b)

0  33  66  100 

100

Coumarin102 Release (%)

a) Emission intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 29

80

100 80

60

60

40

40

20

20

0

0

5

10

15 day

20

25

30

0

Figure 6. a) Time-dependent reduction in the intensity of the fluorescence of coumarin 102 observed for an aqueous 0.1 mg/mL p(DMA-b-PVHC) micellar solution in HEPES buffer (pH 7.4) with encapsulated coumarin 102 as a consequence of the addition of Cu(II) ions to the micellar solution to a final metal ion concentration of 100 μM. b) Timedependent coumarin 102 release profiles obtained following the addition of Cu(II) ions to


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different final concentrations obtained from normalized fluorescence intensities based on the emission maximum of each curve in spectra. c) Encapsulated dyes within the hydrophobic core of p(DMA-b-PVHC) micelles right after micellization (middle), after 1 month in the absence of Cu(II) ions (35% of dye leakage observed, left), and the complete release of encapsulated dyes after 1 month in the presence of 100 μM of Cu(II) ions (right). Each photograph of the left and right vial was taken under ambient and UV light (365 nm), respectively.

Having examined the swelling behavior of core-crosslinked p(DMA-b-PVHC) micelles induced by Cu(II) ion complexation, we attempted to evaluate the effect that the gradual complexation of Cu(II) ions by p(DMA-b-PVHC) micelles had on the sustained release of the hydrophobic model drug coumarin 102. In order to achieve the incorporation of coumarin 102 into the core of p(DMA-b-PVHC) micelles, 3.0 mg of p(DMA-b-PVHC) and 1.0 mg of coumarin 102 were dissolved in 1 ml of THF. To the resulting solution were added dropwise 30 mL of water (HEPES buffer, pH 7.4) over a 12-h period. The Cu(II)-ion-induced sustained release of encapsulated coumarin 102 was studied by fluorescence spectroscopy. The spectrum of the initial micellar solution had the typical strong emission features of coumarin 102, with an emission maximum at 490 nm. After addition to the micelle solution of Cu(II) ions to a final concentration of 100 μM, however, the emission peaks associated with the presence of coumarin 102 decreased steadily in intensity over time. Approximately 7.5% and 25% of coumarin 102 was released from the micelles after 1 h and 1 day, respectively (Figure 6a). Interestingly, the amount of dye released gradually increased in a sustained manner over time, with about 97% of dye being released after 1 month. The release kinetics were further


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investigated changing the final concentration of Cu(II) ions (to 33 μM and 66 μM) and conducting control experiments in parallel whereby no Cu(II) ions were added to the micelle solutions (Figure 6b and S3). Notably, in the absence of Cu(II) ions, the amount of released dye was less than 3% after 1 day, and it reached up to 35% after 1 month. This level of leakage of a hydrophobic dye from polymeric micelles in water is viable, suggesting that p(DMA-b-PVHC) micelles are moderately stable. When Cu(II) ions to a final concentration of 33 or 66 μM were added to the micelle solution, a slower rate of release of the encapsulated coumarin 102 was observed than it was the case when Cu(II) ions were added to a final concentration of 100 μM. The release kinetics, however, still followed a sustained fashion. An intense fluorescence was observed in the photograph of the initially colorless p(DMA-b-PVHC) micellar solution comprising encapsulated coumarin. After 1 month, the solution was still colorless, but it displayed a diminished fluorescence intensity due to the leakage of encapsulated coumarin 102 from the polymeric micelles. This result reflects the observation mentioned above that 35% of dye was released from the micelles over this period of time. After the addition of Cu(II) ions to a final concentration of 100 μM to the solution of micelles comprising encapsulated coumarin, however, the originally colorless solution acquired a yellow color, and it displayed no fluorescence emission, indicating that an almost quantitative release of coumarin 102 had been achieved. Although it took just 1 day to complete the detection of Cu(II) ions (i.e., the completion of metal ion complexation), as indicated by the full swelling of core-crosslinked p(DMA-b-PVHC) micelles, the sustained release of the encapsulated dye continued for 1 month. Although we cannot be entirely certain of the mechanism underlying this prolonged dye release behavior, it is reasonable to assume


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that, unlike a burst release driven by micelle disruption, a diffusion-controlled release from a crosslinked swollen core is a slow, gradual process.

Conclusions A well-defined amphiphilic block copolymer, p(DMA-b-PVHC), was successfully synthesized via RAFT polymerization, followed by post-polymerization modification with 4-phenylthiosemicarbazide. The sensing of Cu(II) ions in aqueous media as described in the present study relies on the slow penetration of Cu(II) ions into the hydrophobic core of PVHC units within p(DMA-b-PVHC) micelles. DLS and AFM data revealed that the size of the micelles increased after the addition of Cu(II) ions, as a consequence of the swelling of crosslinked ionic cores generated by the complexation of Cu(II) ions. The sustained release of the hydrophobic model drug coumarin 102 was achieved by virtue of slow swelling of core-crosslinked p(DMA-b-PVHC) micelles. Overall, the ability of p(DMA-b-PVHC) micelles to detect Cu(II) ions selectively, combined with these micelles’ ability to release a drug in a sustained manner, suggests that this system holds significant promise for the development of therapeutic vehicles with diagnostic capabilities.

Acknowledgments This work was supported by the Basic Science Research Program (NRF2017R1A2B4003861) administered by the National Research Foundation of Korea, funded by the Ministry of Science, ICT, and Future Planning of Korea.


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Graphical abstract


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Use of Core-Cross-Linked Polymeric Micelles Induced by the

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