Amphiphilic TEMPO-Functionalized Block Copolymers: Synthesis, Self

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Amphiphilic TEMPO-Functionalized Block Copolymers: Synthesis, Self-Assembly and Redox-Responsive Disassembly Behavior, and Potential Application in Triggered Drug Delivery Xianbo Shen, Shixiong Cao, Qi Zhang, Jinchao Zhang, Jianli Wang, and Zhibin Ye ACS Appl. Polym. Mater., Just Accepted Manuscript • DOI: 10.1021/acsapm.9b00293 • Publication Date (Web): 24 Jul 2019 Downloaded from pubs.acs.org on July 29, 2019

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ACS Applied Polymer Materials

Amphiphilic TEMPO-Functionalized Block Copolymers: Synthesis, Self-Assembly and Redox-Responsive Disassembly Behavior, and Potential Application in Triggered Drug Delivery

Xianbo Shen,a Shixiong Cao,a Qi Zhang,a Jinchao Zhang,b Jianli Wang,a,* and Zhibin Ye c,* a

College of Chemical Engineering, Zhejiang University of Technology, Hangzhou 310014, P. R. China.

b

College of Chemistry & Environmental Science, Hebei University, Baoding, 071002, P. R. China.

c

Department of Chemical and Materials Engineering, Concordia University, Montreal, Quebec H3G 1M8, Canada.

* Corresponding authors. E-mail: [email protected] (J.W.); [email protected] (Z. Y.)

ABSTRACT:

A

novel

range

2,2,6,6-tetramethylpiperidine-1-oxyl

of

(TEMPO)

amphiphilic functionalized

redox-responsive diblock

copolymers

poly(ethyleneglycol)-b-poly(2,2,6,6-tetramethylpiperidine-1-oxyl-4-ylmethacrylate-co-N-isopropyl acrylamide [PEG-b-P(TMA-co-NIPAM)] has been synthesized in this work via reversible addition-fragmentation chain transfer (RAFT) polymerization followed with functionality transformation. With the possession of the hydrophilic PEG block and the hydrophobic TEMPO containing block, these amphiphilic diblock polymers can conveniently 1

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self-assemble to form stable uniformly sized radical-containing nanoparticles (RNPs), which can thus act as nano-vessels to carry drugs. More interestingly, the self-assembled RNPs exhibit the unique reduction-responsive disassembly upon addition of vitamin C (VC) as a representative reducing agent due to the drastic drop in the surface hydrophobicity following reduction of the TEMPO units. In the proof-of-concept experiments, the VC-triggered release of the encapsulated R6G dye as the model payload from the RNPs at controllable rates has been successfully demonstrated. Meanwhile, the cytotoxicity study with CCK-8 assay confirms the TEMPO-containing block copolymers and their reduced ones are non-toxic at a concentration up to 500 μg mL-1. This series of amphiphilic diblock copolymers is thus highly promising for potential application in triggered drug delivery.

Keywords: Amphiphilic copolymers, redox-responsive behavior, TEMPO, nano-vessels, triggered drug delivery

1. Introduction Stimulus-responsive polymers can undergo programmed reversible changes when exposed to external stimuli, such as temperature,1,2 pH,3 redox,4 light,5 salt,6 sugar,7 and carbon dioxide8, thus rendering valuable applications in diverse fields. Among various stimulus-responsive polymers, redox responsive polymers have attracted wide attention due to their great potential for applications in the fields of catalysis,9-14 energy,15 and medicine.16 Redox responsive polymers can be divided into reduction-responsive polymers and oxidation-responsive polymers. For reduction-responsive polymers, disulfide and diselenide bonds are the most

2


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common active moieties for rendering the reduction responsiveness. Lu et al. reported the use of the disulfide bond as a redox-sensitive linkage for controlled drug release.17 Huang et al. reported that selenium-containing macrocyclic amphiphiles self-assembled in water to form vesicles and the vesicles disassembled upon the addition of excess Vitamin C (VC).18 For oxidation-responsive polymers, thioether-, selenide- or ferrocene-containing moieties are usually used as the active groups. Zhang et al. reported H2O2-responsive thioether-containing polycarbonate copolymers and demonstrated that thioether groups could be oxidized to sulfoxide or sulfone moieties, which led to changes in the polymer hydrophobicity.19 Harada designed a supramolecular responsive hydrogel with self-healing properties through the host-guest interactions of cyclodextrin (as a host) and polyacrylic acid containing ferrocene groups (as a guest). They found that the oxidation state of ferrocene played an important role in the property of hydrogels.20

All examples demonstrated above are focused on the introduction of different redox-responsive groups for endowing the polymers with corresponding properties, which are relatively complex to design and synthesize. Meanwhile, only few studies21 thus far have reported that one single group possesses dual responsiveness towards both oxidation and reduction

at

the

same

time.

Among

existing

redox-active

moieties,

2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO), a typical nitroxide radical, is highly stable under atmospheric conditions. It can be easily oxidized or reduced at mild reaction conditions.22 This property forms the foundation upon which TEMPO can be used as an oxidation catalyst in organic synthesis, as magnetic resonance imaging agent, and as a new

3



flexible battery material.23-25

Nanoparticle-based drug delivery systems, such as dendrimers,26 carbon nanotubes,27 polymeric micelles,28 gold nanoparticles,29 and liposomes,30 have been widely investigated for medical applications. Notably, many studies have focused on the design of reduction-sensitive nanocarriers, in view of the drastic difference in the concentration of the reductive substance between the normal cells and cancer cells.31 The difference endows the targeted release of antitumor drugs. Therefore, increasing attention has been paid to reduction-sensitive materials as drug carriers.32 However, as far as we know, there are only few studies33 reported to date on redox-responsive nanoparticles constructed from radical-containing block polymers as the building materials through self-assembly. Such radical-containing nanoparticles (RNPs) have been found effective in improving cell viability and biofunctions.34 Accordingly, RNPs have the potential for applications in both controlled drug release systems and the treatment of oxidative stress injuries. In addition, these RNPs offer the drug delivery system a detectable property in organisms. With their incorporation in drug delivery systems, the drug delivery routes can be monitored through their radical probes.

Herein, we report the synthesis of a novel range of redox-responsive TEMPO-functionalized amphiphilic block copolymers, poly(ethyleneglycol)-b-poly(2,2,6,6-tetramethylpiperidine -1-oxyl-4-yl methacrylate-co- N-isopropyl acrylamide [PEG-b-P(TMA-co-NIPAM)], which contains a hydrophilic polyethyleneglycol block and a redox-responsive TEMPO-grafted hydrophobic copolymer block. The block copolymers can self-assemble into RNPs in

4


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aqueous solution, which can undergo a valuable redox-responsive disassembly. This assembly/disassembly behavior has been demonstrated and studied with the use of various techniques, including light scattering (DLS), UV-vis spectrophotometry, and transmission electron

microscopy

(TEM).

Finally,

CCK-8

assays

have

indicated

that

the

TEMPO-containing amphiphilic block copolymers and their reduced products are non-toxic at a concentration up to 500 ug·mL-1. Thus, the outstanding redox responsiveness and excellent biocompatibility promote this range of radical-containing block copolymers as promising materials for controllable drug delivery and treatment of oxidative stress injuries.

2. Materials and methods 2.1 Materials 4-Cyan-4-(dodecylsulfanylthiocarbonyl)

sulfanylpentanoic

1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide 2,2,6,6-tetramethylpiperidine-1-oxyl-4-yl

acid

hydrochloride

methacrylate

(DTTCP,

>99%),

(EDC,

>99%),

(TMPM,

>99%),

4-(dimethylamino)-pyridine (DMAP, >99%), sodium tungstate dihydrate (Na2WO4·2H2O, >99%), hydrogen peroxide (H2O2, 30 wt%), ethylene diamine tetraacetic acid (EDTA, >99%), polyethylene glycol monomethyl ethers (mPEG-2000), Rhodamine 6G (R6G, >99%) and Vitamin C (VC, >99%) were purchased from Aladdin. 2,2'-Azoisobutyronitrile (AIBN) was purified by recrystallization from ethanol. N-isopropyl acrylamide (NIPAM) was purified through recrystallization from its solution in mixed n-hexane/toluene (volume ratio: 20:3). All other chemicals were used as received without further purification. 2.2 Synthesis of macromolecular RAFT agent (PEG-DTTCP) (1)

5



In a 250 mL round bottom flask, mPEG-2000 (2.0 g, 1.0 mmol), DTTCP (0.8 g, 2.0 mmol), DMAP (0.122 g, 1.0 mmol), and DCM (100 mL) were added. The mixture was dissolved and cooled in an ice-water bath. Next, a 30 mL DCM solution of EDC (0.4 g, 2.0 mmol) was added dropwise into the solution. Then, the ice-water bath was removed and the solution was stirred for 18 h followed by concentration with a rotary evaporator. The polymer solution was precipitated with 40 mL (×3) of cold ether and the resulting yellow precipitate was isolated by centrifugation. After the repeated precipitation, the yellow product was dried under vacuum at 40 °C. Yield: 83 wt%. 2.3 Synthesis of PEG-b-P(TMPM-co-NIPAM) (2) Diblock copolymer PEG-b-P(TMPM-co-NIPAM) was synthesized by RAFT polymerization. The following ratios among the macromolecular RAFT agent 1 (0.24 g, 0.1 mmol), TMPM, NIPAM, and AIBN as the initiator (I; 5.5 mg, 0.03 mmol) were used to obtain TMPM and NIPAM blocks of different molecular weights: [TMPM]:[NIPAM]:[1]:[I] = 10:90:1:0.33, 20:80:1:0.33, and 30:70:1:0.33. The above components were dissolved in 14 mL of 1,4-dioxane. The solution was stirred under nitrogen for 40 min. The polymerization was then conducted at 70 °C. After 24 h, the polymer solution was precipitated with 40 mL (×3) of cold petroleum ether. The resulting white precipitate was isolated by decanting and then dried under vacuum at 40 °C. 2.4 Synthesis of PEG-b-P(TMA-co-NIPAM) (3) by oxidation of PEG-b-P(TMPM-coNIPAM) A typical procedure is as follows. A block copolymer sample (PEG45-b-P(TMPM18-coNIPAM79) in Table 1; 1 g, containing 1.26 mmol of amine functionality, 1 eq.) was added

6


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into a 50 mL round bottom flask, along with Na2WO4 ·2H2O (103.8 mg, 0.32 mmol, 0.25 eq.), EDTA (55.2 mg, 0.19 mmol, 0.15 eq.), H2O2 (1.29 mL, 12.6 mmol, 10 eq.), and methanol (14 mL). The suspension was stirred at 60 °C for 24 h. Afterwards, the solid was removed by filtration and the residue was precipitated in 40 mL (×3) of cold ether. The resulting orange powder was dried overnight under vacuum at 40 °C. Yield: 72%. 2.5 Preparation of RNPs by polymer self-assembly A typical procedure is as follows. An oxidized polymer sample (the oxidation product of PEG45-b-P(TMPM18-co-NIPAM79) in Table 1, 20 mg) was dissolved in 1 mL of methanol. The solution was added dropwise into 5 mL of water under ultrasonication. Then, methanol was removed through dialysis with a 3500 Da molecular weight cutoff membrane in deionized water for 24 h, rendering the RNP suspension concentration of 2.7 mg/mL. 2.6 Preparation of R6G-loaded RNPs by polymer self-assembly A typical procedure is as follows. An oxidized polymer sample (the oxidation product of PEG45-b-P(TMPM18-co-NIPAM79) in Table 1, 20 mg) and 1 mg of R6G was dissolved in 1 mL of methanol. The solution was added dropwise into 5 mL of water under ultrasonication. Then, methanol and unloaded R6G was removed through dialysis with a 3500 Da molecular weight cutoff membrane in deionized water for 24 h, rendering the suspension of R6G-loaded RNPs with a R6G encapsulation efficiency of 10.3%. 2.7 Reduction-responsive behavior of polymer nanoparticles To study the reduction-responsive behavior of the polymer nanoparticles formed by self-assembly, the changes in particle size, morphology and spectrophotometric transmittance were monitored with DLS, TEM and UV-Vis techniques, respectively, under different

7



Page 8 of 30

concentrations of VC. In specific, 1 mL of RNP suspension was added into a 3.5 mL quartz cell. Next, 1 mL of VC solution was injected into the RNP suspension and the corresponding characterizations were performed. 2.8 In vitro cytotoxicity of PEG-b-P(TMA-co-NIPAM) and PEG-b-P(TMHA-coNIPAM) The cytotoxicity of PEG-b-P(TMA-co-NIPAM) polymers and their reduced products PEG-b-P(TMHA-co-NIPAM) towards the HL-7702 cells (normal hepatic cells) and HELA cells (cancer cells), respectively, was assessed by applying a Cell Counting Kit-8 (CCK-8) assay. The 100 μL logarithmic growth phase cells (5×104 cells mL-1) in the RP1640 medium were transferred to 96-well plates with 6 duplicated wells at each experimental condition, which were incubated for 12 h. Next, the supernatant was replaced with 100 μL of the PEG-b-P(TMA18-co-NIPAM79) or PEG-b-P(TMHA18-co-NIPAM79) (reduction product by VC) solution in fresh RP1640 medium at different concentrations (50, 100, 150, 200, 250 and 500 μg/mL; stabilization for 48 h). The wells without the addition of any polymer were served as the controls. Afterwards, the supernatant was replaced again with 100 μL of the fresh RP1640 medium containing CCK-8 and was incubated for 2 h. The optical density (OD) was measured at 450 nm by using the microplate reader. The cell viability was calculated according to the following equation: Cell viability (%) = (ODsample)/(ODcontrol) × 100%

(1)

where ODsample and ODcontrol represent the values from the sample and control wells, respectively. 2.9 Characterizations

8


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1H

NMR spectra were obtained on a 500 MHz NMR spectrometer (Bruker AV500). The

number-average molecular weight (Mn) and weight-average molecular weight (Mw) of PEG-b-P(TMPM-co-NIPAM) and PEG-b-P(TMA-co-NIPAM) were determined with a size exclusion chromatography (SEC) system incorporating a Waters 1525 HPLC pump, a Styragel® HR2 column , and a Waters 2414 refractive index detector. DMF (1.0 mL·min-1) was used as the mobile phase. Fourier-transform infrared (FTIR) spectroscopic analyses were conducted on a BRUKER TENSOR Ⅱ spectrometer. Samples were prepared with KBr pellets and the spectral resolution was 4 cm-1 and 64 scans were performed on each sample. Static water contact angle measurements were conducted to test the hydrophilicity of the polymers using a sessile drop method on a Data Physics OCA-20 instrument. The average particle size and distribution of the self-assembled polymer nanoparticles were determined with dynamic light scattering (DLS) on a Malvern Instruments Zetasizer Nano ZS90 at 25 °C. Transmission electron microscopy (TEM) characterizations of the morphology of the polymer nanoparticles were conducted on a FEI Titan-G2 80-200 chemi STEM instrument. The UV-Vis measurements were performed on an Agilent Cary 60 spectrophotometer at 525 nm. The cyclic voltammetry (CV) tests were performed on a CHI 660E electrochemical working station within a potential range from 0 to 1.5 V at a scanning rate of 50 mV/s.

3. Results and Discussion 3.1 Synthesis and Characterizations of PEG-DTTCP (1) As shown in Scheme 1, the macromolecular RAFT agent, PEG-DTTCP (1), was synthesized via esterification of hydroxyl-terminated PEG with DTTCP (the thioester containing

9



Page 10 of 30

carboxylic acid) in the presence of EDC and DMAP. Following the synthesis, it was characterized with 1H NMR spectroscopy to verify its chemical structure and thioester end functionality. In the 1H NMR spectrum (see Figure S1 in Supporting Information), the peaks (f, g, h) within 1.73−0.85 ppm are assigned to methylene and methyl protons on the pendant alkyl group in the thioester chain transfer agent. The signals at δ = 3.38 ppm (e, −S-CH2−), δ = 2.72.3 ppm (c, −C(O)−CH2−CH2−), and δ = 1.9 ppm (d, −(CN)C(CH3)−) also arise from the corresponding protons in the thioester chain transfer agent. The signals within 3.823.46 ppm arise from the methylene protons of the PEG repeat units. The spectrum of PEG-DTTCP agrees well with the predicted structure. Scheme 1. Synthesis route of PEG-b-P(TMA-co-NIPAM). O

O O

S

OH + HO 45

S S

NC

DCC, DMAP, DCM C12H25

O

RT

S

O

45

S

C12H25

S

NC

1 NIPAM / TMPM O O

O S

O

m

45

CN

3

O

N

O O

S

n

NH

O

C12H25

S

O

45

S

H2O2/NaWO4/EDTA MeOH/60 oC/24 h

O

m

CN

O

O O

N

S

n

NH

C12H25

S

2

H

3.2 Synthesis and Characterization of PEG-b-P(TMPM-co-NIPAM) (2) With PEG-DTTCP as the macromolecular RAFT agent, PEG-b-P(TMPM-co-NIPAM) diblock copolymers have been synthesized by RAFT copolymerization of NIPAM and TMPM (see Scheme 1). The polymerizations were carried out at three different molar feed ratios of TMPM to NIPAM (10:90, 20:80, and 30:70), rendering three diblock copolymers with the P(TMPM-co-NIPAM) block of different compositions (PEG45-b-P(TMPM8-co10


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NIPAM90),

PEG45-b-P(TMPM18-co-NIPAM79),

PEG45-b-P(TMPM27-co-NIPAM68),

respectively; see Table 1). Full conversions of both monomers were reached in the polymerization runs with the absence of residue monomers in the polymerization solution as per 1H NMR characterization. a

b O O O 45 PEG

O

c c

e

f

l m

d CN j k

O

O g hi

m S

S

n

O NH

q p

s r

9

S

n o

N H

Figure 1. 1H NMR spectra of diblock PEG-b-P(TMPM-co-NIPAM) (in CDCl3) synthesized at different TMPM/NIPAM monomer molar feed ratios of (A)30:70, (B) 20:80, and (C) 10:90.

Figure 1 shows the 1H NMR spectra of the three diblock copolymers synthesized at the different DMPM/NIPAM feed ratios, with characteristic proton signals of both TMPM and NIPAM repeat units observed. In specific, a characteristic signal (g) of the TMPM repeat unit at δ = 5.06 ppm is observed, corresponding to the methine proton (−(CH2)CH(CH2)−OC(O)−) in the pendant piperidine group. A characteristic signal (n) of NIPAM units at δ = 3.97 ppm is observed, typical of methine protons in the pendant group. With these characteristic peaks, 11



Page 12 of 30

the composition of the DMPM-NIPAM copolymer block has been determined. Based on the end group analysis, the number average molecular weight Mn, NMR of the diblock copolymers has also been calculated from the 1H NMR spectra according to the following equation: Mn, NMR = [(Ig)/(Ia/3)] × 225 + [(In)/(Ia/3)] × 113 +2385

(2)

where Ig and In are the peak integral values of peak g at δ = 5.06 ppm and peak n at δ = 3.97 ppm, respectively. The values of 225, 113, 2385 correspond to the molecular weights (g mol-1) of the TMPM unit, NIPAM unit, and PEG-DTTCP, respectively. The Mn,NMR data are summarized in Table 1, along with the theoretical Mn data calculated based on full conversions of both monomers. Two sets of absolute Mn data agree very well, also confirming the full conversions of both monomers in the RAFT polymerization. The copolymerisation parameters of NIPAM and TMPM are showed in Table 1. It has been calculated according to the following equation: r = [(n(TMPM)test)/(n(TMPM)theory)]/[(n(NIPAM)test)/(n(NIPAM)theory)]

(3)

Where n(TMPM)test and n(TMPM)theory are polymerization degree of TMPM for test and theory, respectively. Where n(NIPAM)test and n(NIPAM)theory are polymerization degree of NIPAM for test and theory, respectively. The average molecular weights and molecular weight distribution of the three PEG-b-P(TMPM-co-NIPAM) diblock copolymers were also determined with SEC (see Figure S2 in Supporting Information for SEC elution curves). Monomodal SEC curves are observed. Relative to the curve of the macromolecular RAFT agent, the elution peaks of the diblock copolymers are left-shifted with significantly enhanced elution time, but with similar peak width and shape. The diblock copolymers are featured with low PDI values (1.121.17),

12


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indicative of their narrow molecular weight distributions arising from “living” characteristics of the RAFT polymerization. The number-average molecular weight and polydispersity index data determined with SEC are also summarized in Table 1.

Table 1. Synthesis of PEG-b-P(TMPM-co-NIPAM)

No

Mn a

Sample

(theory) 1 2

PEG45-b-P(TMPM8-co-NIPAM90) PEG45-b-P(TMPM18-co-NIPAM79

Mn b (1H

NMR)

Mn c

PDI c

(SEC)

(SEC)

rd

14800

14350

14590

1.17

0.80

15920

15360

14950

1.14

0.91

17050

16150

15460

1.12

0.93

) 3

PEG45-b-P(TMPM27-co-NIPAM68 )

[C]0 represent the initial concentration. a Mn calculated according to Mn = ([M1]0/[I]0 × 113) + ([M2]0/[I]0 × 225) + 2385, where [M1]0, [M2]0 and [I]0 are the feed moles for TMPM, NIPAM and PEG-DTTCP, respectively. b Mn calculated via 1H NMR spectroscopy according to Eq. 2. c Mn and PDI determined with SEC relative to PS standards. r d represent the copolymerisation parameters of NIPAM and TMPM, which were calculated from the Eq. 3.

3.3 Synthesis and Characterization of PEG-b-P(TMA-co-NIPAM) (3) Oxidation of the TMPM units in PEG-b-P(TMPM-co-NIPAM) block polymers was carried out with hydrogen peroxide as the oxidant and EDTA/Na2WO4 as the catalyst at 60 °C to render TEMPO radical-containing PEG-b-P(TMA-co-NIPAM) diblock polymers. After oxidation of PEG-b-P(TMPM-co-NIPAM), the color of the sample changed to orange (see Figure S3 in Supporting Information) . It can be interpreted as TEMPO color. Because of the

13



disturbance of TEMPO radicals, the

1H

Page 14 of 30

NMR characterization of the resulting

PEG-b-P(TMA18-co-NIPAM79) block polymers was performed in the presence of a small amount of phenylhydrazine to reduce nitroxide radical to hydroxylamine.35 O

O PEG 45

O

S

O CN

18

O

O

S

79

O NH

9

S

N OH

Figure 2. A comparison between the1H NMR spectrum of PEG-b-P(TMA18-co-NIPAM79) in the presence of phenylhydrazine in DMSO and PEG-b-P(TMPM18-co-NIPAM79) in CDCl3.

The 1H NMR spectrum of PEG-b-P(TMA18-co-NIPAM79) (see Figure 2) shows that the characteristic peaks of the PEG, TMPM and NIPAM repeated units are well retained with almost no change compared with those of the PEG-b-P(TMPM18-co-NIPAM79). The broad signal within 7.12−7.24 ppm corresponds to the hydroxyl group generated from the reduction of TEMPO to TEMPOH. SEC was used to estimate average molecular weights of PEG-b-P(TMA-co-NIPAM) diblock copolymers (see Figure S4 in Supporting Information for SEC elution curves). The monomodal elution curves are retained with slightly increased 14


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Mn values for the oxidation products PEG-b-P(TMA-co-NIPAM).

Figure

3.

FTIR

spectra

of

PEG-DTTCP,

PEG-b-P(TMPM18-co-NIPAM79),

and

PEG-b-P(TMA18-co-NIPAM79).

FTIR spectra of PEG-DTTCP, PEG-b-P(TMPM18-co-NIPAM79) and PEG-b-P(TMA18-coNIPAM79) are shown in Figure 3. The wide peak of 3250-3750 originated from the stretching vibration of -OH in PEG for FTIR spectra of PEG. When NIPAM and TMPM monomers are polymerized on PEG chains, the broad peaks of 3250-3750 were not only from the stretching vibration of -OH in PEG, but also from the stretching vibration of N-H in NIPAM and TMPM. Because the content of NIPAM in PEG-b-P(TMPM18-co-NIPAM79) was higher than that in TMPM, even if TMPM N-H is completely converted to N-O, it has little effect on its peak pattern, so no obvious change of peak pattern can be found in 3250-3750. After oxidation, another new peak, assigned to the N-O• stretching vibration, appears at 1322 cm-1 in the spectrum of PEG-b-P(TMA18-co-NIPAM79), confirming the successful oxidation and the presence of TEMPO radicals in PEG-b-P(TMA18-co-NIPAM79). The redox property of PEG-b-P(TMA18-co-NIPAM79) has been characterized with

15



cyclic voltammetry (see Figure 4). A pair of reversible redox peaks with narrow peak separation is found near 0.85 V (vs. Ag/AgCl). It was reported that NIPAM-containing block copolymers with TEMPO side groups had an oxidation potential of 0.81 V versus AgCl/Ag (anodic peak), which is in good agreement with the result herein.36 The redox peaks correspond to the reversible switch between the nitroxide function and the oxoammonium cation, confirming the reversible redox property of PEG-b-P(TMA18-co-NIPAM79).

Figure 4. Cyclic voltammetry of PEG-b-P(TMA18-co-NIPAM79) at 50 mV s−1 (3 cycles with 3 mg of PEG-b-P(TMA18-co-NIPAM79) and 10 mmol of Bu4NClO4 in 10 mL of DCM).

Figure 5. Water contact angles of PEG-b-P(TMPM18-co-NIPAM79) (a), PEG-b-P(TMA18-co-NIPAM79) (b), PEG-b-P(TMHA18-co-NIPAM79) (c) and PEG-b-P(TMA+18-co-NIPAM79) (d).

16


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With their possession of the TEMPO units with reversible redox property, we have investigated the surface wetting properties of diblock copolymers and the effects of reduction/oxidation on the properties. For this purpose, PEG-b-P(TMA18-co-NIPAM79) was reduced to PEG-b-P(TMHA18-co-NIPAM79) with VC (a typical reducing agent reducing

nitroxide

radical

to

PEG-b-P(TMA+18-co-NIPAM79)

with

hydroxylamine), NaClO

(nitroxide

and radical

oxidized oxidized

to to

oxoammonium cation). Water contact angle (CA) measurements were undertaken on the surfaces of PEG-b-P(TMPM18-co-NIPAM79), PEG-b-P(TMA18-co-NIPAM79), PEG-b-P(TMHA18-co-NIPAM79), and PEG-b-P(TMA+18-co-NIPAM79) as shown in Figure 5. The CA is about 50° (Figure 5a) and 88° (Figure 5b) for PEG-b-P(TMPM18-co-NIPAM79) and PEG-b-P(TMA18-co-NIPAM79), respectively. For the reduced product and oxidized product, the corresponding CA becomes about 43° (Figure 5c), 33° (Figure 5d), respectively.37,38 On one hand, the CA data reveals that the TMPM-containing polymer becomes more hydrophobic after the transformation of tertiary amine (N-H) groups to nitroxide radicals (N-O·). This facilitates the preparation of polymer nanoparticles by self-assembly of the amphiphilic block copolymers. On the other hand, the hydrophilicity of the nitroxide radical-containing polymers can be greatly improved by reduction of the nitroxide radicals (N-O·) to hydroxylamine (N-OH) groups or oxidation to oxoammonium cations (N+=O). This endows the nitroxide radical-containing polymer the unique responsiveness towards the redox stimuli. These combined valuable features allow us to develop the strategy herein to design self-assembled RNPs with redox responsiveness. 3.2 RNPs by Self-Assembly Given the two drastically different constituting blocks, RNPs are easily prepared by self-assembly of the amphiphilic PEG-b-P(TMA-co-NIPAM) diblock copolymers. Herein, we demonstrate the synthesis of RNPs and study their properties with the use of one of the diblock copolymers, PEG-b-P(TMA18-co-NIPAM79). For the self-assembly, a methanol solution of PEG-b-P(TMA18-co-NIPAM79) was added 17



simply into water, followed by the removal of methanol. The resulting RNPs were characterized with DLS and TEM. Figure 6a shows DLS particle size distribution of the RNPs, revealing a single distribution with a mean hydrodynamic size of 180 nm (see Figure 6a). Figures 6b and 6c show the TEM images of the RNPs, revealing their uniform nanosphere morphology with an average diameter of about 50 nm. As also seen from the images, some of the RNPs aggregate to form larger agglomerates under TEM. The difference in the sizes measured with the two techniques should be due to the fact that DLS gives the larger hydrodynamic sizes in aqueous suspension.28 These results confirm the successful self-assembly of the diblock copolymer to form RNPs.

Figure 6. (a) Intensity-average size distribution of RNPs from DLS; (b and c) TEM images of the self-assembled RNPs prepared from PEG-b-P(TMA18-co-NIPAM79).

3.3 Redox-Responsive Behavior of RNPs It has been shown that nitroxide radicals can be easily reduced and oxidized to produce hydrophilic hydroxylamine and oxoammonium cation, respectively.39-41 Along with the above CA results, we thus reason that these RNPs should have the redox-responsive behavior. Through an initial experiment, we have found that the above self-assembled nanoparticles dissolve upon the addition of excess VC (a typical

18


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reducing agent), with the originally opaque dispersion becoming clear and transparent (see Figure S5 in Supporting Information). To quantitatively evaluate the redox-responsive dissolution, we have applied UV-vis spectrophotometry to monitor the changes in the absorption of the dispersion of RNPs in response to VC. As shown in Figure 7, the absorbance of the dispersion at 530 nm exhibits gradual decreases upon the addition of VC at different molar equivalent relative to the TEMPO units, accompanied with the simultaneous change of the aqueous dispersion from turbid to transparent. This suggests the dissolution of the RNPs upon the addition of VC. Meanwhile, the absorbance drop shows a strong rate-dependence on VC concentration, but with almost the same ultimate absorbance obtained at the different VC dosages. This VC-triggered dissolution of RNPs should result from the transformation of the hydrophobic nitroxide groups to hydrophilic hydroxylamine groups, which leads to the disassembly of the RNPs and in turn the increase in transmittance. The addition of VC at a higher dosage leads to a faster dissolution of RNPs, which offers a strategy for rate control.

Figure 7. Absorbance of RNPs at 530 nm upon the addition of VC at different molar equivalents relative to TEMPO. 19



Figure 8 shows the TEM images of the reduction product obtained after the reduction with 40 molar equivalent VC for 10 min. The uniform nanosphere morphology no longer exists. Instead some random polymer structures are observed under TEM, further confirming the disassembly of RNPs upon reduction.

Figure 8. TEM image of the reduced product of the RNPs upon reduction with 40 equivalent VC for 10 min.

To further reveal the mechanistic process of the VC-triggered disassembly of the RNPs, we have employed DLS to monitor the changes in the measured hydrodynamic size and the counter rate of the dispersion upon the addition of VC at different dosages (see Figure 9). From Figure 9(a), the measured size based on intensity of the dispersion gradually increases under the various VC dosages and then reaches a plateau. However, the count rate shows an initial increase and then decreases in all the cases. In particular, the rates of both the initial increase and the latter decrease show strong dependences on the VC dosage, with higher rates observed at the increasing VC dosages. Because the count rate has a positive relevance with the number and size of nanoparticles, this trend of change in the counter rate reveals that the disassembly of the RNPs triggered by VC addition is comprised of two steps.42 The first step is the swelling of RNPs by water due to the gradual reduction of the nitroxide groups and the 20


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increase in the hydrophilicity of the nitroxide-containing polymer block. The swelling results in the increases of the size of RNPs and count rate. The second step is the dissociation of the swollen RNPs after the reduction of a sufficient quantity of nitroxide groups, leading to the decrease in the number of RNPs and in turn the count rate (see Figure 10). The increase in VC dosage leads to the enhanced reduction rate and thus faster swelling and dissociation rates.

Figure 9. DLS results of the RNP dispersion upon the addition of VC at different dosages: (a) size and (b) counter rate.

Swelling

Dissolving

Figure 10. Schematic illustrations of the process of RNPs dispersion upon the addition of VC

In order to further verify that the disassembly of the RNPs results from the reduction of nitroxide radicals by VC rather than from its acidity, we have conducted another control experiment by adding acetic acid into the dispersion to reach the same pH (pH

21



Page 22 of 30

≈ 3) that would be achieved by the addition of 20 molar equivalent of VC. The dispersion was also monitored with UV-vis spectrophotometry. As shown in Figure S6, the absorbance of the dispersion shows little changes over a month. This indicates that the nanoparticles are not sensitive to pH and confirms their reduction responsiveness.

We have also attempted to demonstrate the oxidation-responsive behavior of the RNPs following our reasoning. Surprisingly, flocs appeared when 50 equivalent of NaClO was added into the RNP dispersion (see Figure S7 in Supporting Information). This possibly results from the oxidation of the nitroxide groups in RNPs to hydrophilic oxoammonium cationic polymer and the precipitation of the polymer due to the high concentration of salt in sodium hypochlorite solution.40 With the dissolution and precipitation occurring simultaneously, DLS and UV-vis are not applicable for online monitoring of the change. Nevertheless, the water contact angle data (see Figure 5) of the oxidized PEG-b-P(TMA18-co-NIPAM79) by sodium hypochlorite evidences the oxidation-responsive behavior of RNPs. We will continue to search for more suitable oxidants to render the oxidation-triggered dissolution of these RNPs without causing polymer precipitation in our further work. The unique reduction responsiveness of self-assembled RNPs endows their potential applications in triggered drug delivery. For that purpose, cytotoxicity of the RNPs is investigated. By applying a CCK-8 assay in HL-7702 (normal hepatic cells) and HELA cells (cancer

cells),

the

cell

cytotoxicity

of

PEG-b-P(TMA18-co-NIPAM79)

and

PEG-b-P(TMHA18-co-NIPAM79) has been evaluated. At a series of concentrations (50, 100,

22


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150, 200, 250, and 500 μg·mL−1), both polymers display negligible cell cytotoxicity for HL-7702 with >90% of cell viability (see Figure 11(a)), demonstrating their good biocompatibility. On the contrary, with the HELA cells, the cell viability shows an obvious trend of decrease with the increase of polymer concentration, especially for TEMPO-containing PEG-b-P(TMA18-co-NIPAM79) (see Figure 11(b)). One possible explanation for the latter is the oxidation of TEMPO-containing PEG-b-P(TMA18-coNIPAM79) and TEMPOH-containing PEG-b-P(TMHA18-co-NIPAM79) to the oxoammonium cation-containing PEG-b-P(TMA+18-co-NIPAM79) by high levels of reactive oxygen species present in HELA cells, which inhibits cell growth due to their positive charge. The two polymers are thus demonstrated to show negligible cytotoxic effects on normal cells but inhibit the growth of cancer cells, which is desirable for their applications as redox-responsive nanoreservoirs of anticancer drug delivery.

Figure 11. Cell viability of HL-7702 cells (a) and HELA cells (b) cultured with PEG-b-P(TMA18-co-NIPAM79) and PEG-b-P(TMHA18-co-NIPAM79), respectively.

As a preliminary proof-of-concept demonstration, the VC-triggered payload release from RNPs of PEG-b-P(TMA18-co-NIPAM79) has been shown with the use of R6G dye as a model compound. For the controlled release, R6G was loaded at an encapsulation percentage of 10.3 23



Page 24 of 30

wt%. The VC-triggered release of R6G from the RNPs was monitored with UV-vis. Figure 12 shows the release profiles under the stimulus of VC at three different dosages (10, 15 and 20 equivalent of VC). The increase of VC dosage leads to a faster and a more complete release of R6G from the RNPs due to their triggered disassembly, demonstrating the potential for practical applicability.

Figure 12. In vitro R6G release profiles from PEG-b-P(TMA18-co-NIPAM79) RNPs upon the addition of VC at different dosages.

4. Conclusions A

novel

series

of

amphiphilic

TEMPO

radical-containing

block

copolymers,

PEG-b-P(TMA-co-NIPAM), has been designed and synthesized herein with the use of the RAFT technique. With the hydrophobicity of the TEMPO groups, the block copolymers can uniquely self-assemble into uniform RNPs. Meanwhile, a further added valuable advantage of these novel polymers is their unique VC-responsiveness, which offers an efficient strategy to trigger the responsive dissociation of the self-assembled RNPs due to the reduced hydrophobicity upon reduction. The CCK-8 assay has confirmed the non-cytotoxicity of the amphiphilic block copolymers and their reduced products. Proof-of-concept experiments

24


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have also verified their application as nanoreserviors for VC-triggered release of R6G. With these combined desirable features (including self-assembling capability, outstanding reduction responsiveness, good biocompatibility), these block copolymers and their RNPs have a potential as responsive materials for treatments of cancer and oxidative stress injuries. With the simple synthesis, this design strategy incorporating the valuable TEMPO radicals can be applied to a broad range of stimulus responsive polymers.

ASSOCIATED CONTENT Supporting Information TEM preparation procedure, the VC-triggered release procedure and more characterization results using 1H NMR, GPC, photographs, and solution transmittance measurements.

AUTHOR INFORMATION Notes The authors declare no competing financial interes.

ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (No. 21374103), Zhejiang Provincial Natural Science Foundation of China (LY18B040004).

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TOC

self-assembly in water

ascorbic acid or NaClO

N-O N-OH or

N O

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Amphiphilic TEMPO-Functionalized Block Copolymers: Synthesis, Self

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