Disulfide-Functionalized Unimolecular Micelles as Selective Redox

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Disulfide-Functionalized Unimolecular Micelles as Selective Redox-Responsive Nanocarriers Christian Porsch,a Yuning Zhang,b Maria I. Montañez,a Jani-Markus Malho,c Mauri A. Kostiainen,d Andreas M. Nyström,b and Eva Malmströma,*

a

School of Chemical Science and Engineering, Department of Fibre and Polymer Technology, KTH Royal Institute of Technology, SE-100 44 Stockholm, Sweden. b

IMM Institute of Environmental Medicine, Karolinska Institutet, SE-171 77 Stockholm, Sweden. c

d

Department of Applied Physics, Aalto University, FI-00076 Aalto, Finland.

Biohybrid Materials Group, Department of Biotechnology and Chemical Technology, Aalto University, FI-00076 Aalto, Finland.

KEYWORDS Redox-responsive, self-condensing vinyl polymerization, unimolecular micelle, polymer nanoparticles, drug delivery

ACS Paragon Plus Environment

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ABSTRACT

Redox-sensitive hyperbranched dendritic-linear polymers (HBDLP) were prepared and stabilized individually as unimolecular micelles with diameters in the range 25-40 nm. The high molecular weight (500-950 kDa), core-shell amphiphilic structures were synthesized through a combination of self-condensing vinyl copolymerization (SCVCP) and atom transfer radical polymerization (ATRP). Cleavable disulfide bonds were introduced, either in the backbone, or in pendant groups, of the hyperbranched core of the HBDLPs. By triggered reductive degradation, the HBDLPs showed up to a sevenfold decrease in molecular weight, and the extent of degradation was tuned by the amount of incorporated disulfides. The HBDLP with pendant disulfide-linked functionalities in the hyperbranched core was readily post-functionalized with a hydrophobic dye, as a mimic for a drug. An instant release of the dye was observed as response to a reductive environment similar to the one present intracellularly. The proposed strategy shows a facile route to highly stable unimolecular micelles, which attractively exhibit redox-responsive degradation and cargo release properties.

INTRODUCTION With the rapid emergence of sophisticated and accessible controlled radical polymerization techniques, such as atom transfer radical polymerization (ATRP)1, 2, reversible additionfragmentation chain transfer polymerization (RAFT)3, 4, and nitroxide-mediated polymerization (NMP)4, significant opportunities in terms of macromolecular design have emerged. The unique possibilities in tailoring polymer composition, morphology, molecular weight, and end-group chemistries, from commercially available vinyl monomers, have promoted substantial developments in the entire polymeric materials field. Amphiphilic polymers are macromolecules


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displaying both hydrophobic and hydrophilic characteristics. Such polymer structures have shown to be of great interest since they have the ability to spontaneously self-assemble into discrete structures in the nanometer scale. By varying the composition and architecture of such amphiphilic polymers, assemblies in the shape of micelles, rods, cylinders, and polymersomes have been demonstrated.5,

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Polymer assemblies, especially micelles, have shown particular

utility in drug delivery.7-10 Their core-shell morphology has been proved to facilitate both the dissolution and in vivo delivery of poorly soluble, and highly toxic, therapeutics, and thereby reduce side effects and allow for the administration of higher doses, thus resulting in improved therapeutic efficacy.11-13 Further, since renal elimination is suppressed by increased hydrodynamic volume, polymer micelles can prolong the blood circulation time of a therapeutic14,

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, and consequently improve tumor uptake via the enhanced permeation and

retention (EPR) effect.16, 17 Although polymer amphiphiles may form stable assemblies, and thereby provide benefits during treatment, they are always in equilibrium between their self-assembled micellar state and individual polymer chains. Consequently, there are concerns regarding the micellar stability upon injection and circulation in the blood stream (ca. 5 L). Dilution below the critical micelle concentration (CMC)18, changes in solution conditions, or shear forces, may lead to micelle destabilization and premature therapeutic release, resulting in undesirable systemic toxicity.19, 20 To prevent this, attention has been directed toward the synthesis of so called unimolecular micelles, where a single molecule can exhibit micelle-like properties, first proposed by Newkome et al. in 1985.21 One promising route to form such individually stabilized micelles is the synthesis of amphiphilic, highly branched star-shaped macromolecules of high molecular weight with distinctly different hydrophilicity in the core and the shell. Generally, such


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macromolecular architectures can be synthesized via two main strategies: the arm-first or the core-first approach. In the arm-first approach, one appealing strategy has been to form a self-assembly of amphiphilic polymers followed by a subsequent cross-linking event, covalently stabilizing either the core or the shell of the assembly to form a stabilized macromolecule.22-24 Wooley et al. have extensively reported on shell crosslinked knedel-like (SCK) nanoparticles (NPs) and their use in therapeutic delivery over the past decades.25-27 Another method is the one-pot synthesis of block copolymers with the introduction of a low amount of difunctional monomer during the synthesis of the hydrophobic block to covalently link them together.28-33 Recently, these strategies have been further explored by the formation of the NPs via the polymer-induced self-assembly (PISA) technique.34, 35 In the core-first approach, significant focus has been on the use of dendritic polymers, especially dendrimers/dendrons, due to their globular structure and high number of functional groups in the periphery.36-38 However, the tedious and costly synthesis related to dendrimers, as well as their present restrictions in size (typically ≤15 nm39), have directed attention to their less perfect, hyperbranched, analogues. A significant amount of work has been reported on the synthesis of star-like polymers, comprised of a hyperbranched hydrophobic core decorated with a multitude of linear hydrophilic arms, to form amphiphilic core-shell type polymers40-42, and their applicability as drug delivery systems.43-48 Furthermore, since the introduction of the concept of self-condensing vinyl polymerization (SCVP) by Fréchet et al.49, chain-wise synthesis of hyperbranched homo-and copolymers has attracted attention. Our group, among others, has elaborated on the versatile use of SCVP in the synthesis of high molecular weight, amphiphilic polymers with a hydrophobic hyperbranched core, decorated with a multitude of linear


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hydrophilic arms, to form core-shell type NPs.50-53 Employing a radical polymerization technique, an exceptionally versatile platform to form hyperbranched dendritic-linear macromolecules is achieved. Recently, we demonstrated that linear polymers of oligo(ethylene glycol) methyl ether methacrylate (OEGMA) can stabilize hyperbranched polymers synthesized via SCVCP, and by appropriate tailoring of their architecture and molecular weight form unimolecular micelles.51 One challenge for the applicability of unimolecular micelles as drug delivery systems is their high probability of bioaccumulation related to their inherent high molecular weight.54 Further, the therapeutic delivery from the micelles still relies on physical entrapment in the micellar core where the diffusion controlled release may cause toxicity due to premature release during circulation. Disulfide bonds have been widely explored as selectively cleavable linkers for therapeutic delivery due to the pronounced difference in reduction potential between intra- and extracellular conditions.55,

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Recent studies report how disulfide bonds conveniently can be

introduced to the backbone of hyperbranched polymers by the SCVP technique.50, 57, 58 In this work we are further advancing our previously reported unimolecular micelle platform by exploring the opportunity to functionalize the polymers with disulfide bonds. We are reporting on the synthesis of redox-responsive, hyperbranched dendritic-linear polymers (HBDLP) by the introduction of disulfide bonds either in the hyperbranched backbone to allow for biodegradation, or in the pendant groups to allow for triggered release of cargo. The HBDLP ability to form unimolecular micelles, and selectively degrade or release a covalently attached guest molecule under reductive conditions is evaluated.

EXPERIMENTAL


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Materials All chemicals were obtained from Sigma-Aldrich and used as received if not stated otherwise. Triethylamine (TEA) and TLC silica gel 60 F254 plates were purchased from Merck. Oligo(ethylene glycol) methyl ether methacrylate (OEGMA475, average Mw 475 g mol-1) and hexyl acrylate (HA, Mw 130 g mol-1) were activated by passage through a column of neutral aluminum oxide prior to use. Human breast cancer cell line, MCF-7 was purchased from the American Type Culture Collection (ATCC). The cell line was maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10 % (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg mL-1 streptomycin, and incubated at 37 °C with 5 % CO2. FBS was purchased from Hyclone Laboratories.

Methods Nuclear magnetic resonance (NMR). 1H-, 13C-NMR (1D) spectra for structure analysis were recorded on a Bruker Avance AM 400 NMR instrument using CDCl3 as solvent. The residual solvent peak was used as internal standard if not stated otherwise. Size exclusion chromatography (SEC). SEC measurements were performed on two different instruments. Triple detection GPC was equipped with a GPCmax VE2001 auto-sampler, two Viscotek D6000 columns (and a guard column), and a triple detector array TDA305 (refractive index, light scattering and viscometer) with a mobile phase of DMF containing 0.01 M lithium bromide at 60 °C and a flow-rate of 1 mL min-1. At the time of the analysis the viscometer detector was out of order and only RI and RALLS/LALLS detection was performed. SEC was also performed on a TOSOH EcoSEC HLC-8320GPC system equipped with an EcoSEC RI detector and three columns (PSS PFG 5 µm; Microguard, 100 Å, and 300 Å) (MW resolving


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range: 100-300 000 g mol-1) from PSS GmbH, using DMF (0.2 mL min-1) with 0.01 M LiBr as the mobile phase at 50 °C. A conventional calibration method was created using narrow poly(methyl methacrylate) standards ranging from 700 to 2 000 000 g mol-1, and used to determine the average molar masses (number-average molar mass, Mn and weight-average molar mass, Mw) and the dispersity (ÐM = Mw/Mn). Corrections for flow rate fluctuations were made using toluene as an internal standard. PSS WinGPC Unity software version 7.2 was used to process the data. Dynamic light scattering (DLS). Samples were analyzed with a Malvern Zetasizer NanoZS at 25 °C and 37 °C in PBS with polymer concentrations of 1.25 mg mL-1 if not stated otherwise. Each sample was allowed to equilibrate for 2 minutes prior to analysis and all results are averages of minimum three individual samples where each sample data are an average of 6 measurements. Cryogenic transmission electron microscopy (cryo-TEM). 3 µL of NP dispersion (1.25 mg/mL) was pipetted on to holey carbon copper grids under 100 % humidity and the excess amount of sample was blotted away with filter paper for 1.5–2.0 s. Samples were subsequently plunged into -170 °C ethane/propane mixture. The vitrification was done using Fei Vitrobot Mk3. Vitrified samples were then cryo-transferred to the microscope. The imaging was performed using Jeol’s 3200FSC cryo-transmission electron microscope. The imaging was carried out operating the microscope at 300 kV in bright field mode with an Omega-type Zeroloss energy filter. The specimen temperature was maintained at -187 °C throughout the imaging. Gatan Ultrascan 4000 CCD camera was used to acquire the images.


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Nanoparticle formation. The HBDLPs were formed into nanoparticles employing a direct dissolution method. HBDLP (>10 mg) was dissolved in PBS (1.25 mg mL-1) and allowed to equilibrate for minimum 24 hours on a shaking device (250 rpm) covered with aluminum foil. Degradation of HBMIs by dithiothreitol (DTT). The degradation of the HBMIs (Table 1, entries 1-4) was performed directly in SEC solvent (DMF, 0.01 M LiBr). The HBMIs were dissolved in DMF (1 mL, 3 mg mL-1) in glass vials during stirring overnight. To each vial DTT stock solution (in DMF) was added to ensure a final DTT concentration of 10 mM. For each HBMI a blank sample without DTT was analyzed as control. The solutions were subjected to vacuum (1 min), purged with argon (10 min), and allowed to stir for 24 h at room temperature, covered with aluminum foil. The samples were evaluated by DMF-SEC analysis. Degradation of HBDLPs by DTT/glutathione (GSH). For each HBDLP (Table 2, entries 57) three different DTT concentrations (10 µM, 1 mM, 10 mM), and a control sample in pure PBS were analyzed. For HBDLP(SS100) treatment with 10 mM GSH and 10mM GSH + catalytic amount of TEA was also performed. The HBDLPs were dissolved in PBS (1 mL, 15 mg mL-1) in glass vials for 24 h on a shaking device (250 rpm). To each vial, DTT/GSH stock solution (in PBS) was added (95% conversion, and ended by deactivator addition (Cu(II)Br2) to support carbon-bromine terminated chain-ends. The high conversions favor high molecular weight species since linear or lightly branched chains are consumed in the latter stages of the polymerization. The use of a polar solvent, such as anisole, has shown to

Scheme 1. Schematic illustration of the synthetic route to the unimolecular micelles with a) backbone-cleavable disulfide bonds b) azide-functional cleavable pendant disulfide bonds. (SSx) in the polymer names denotes the molar fraction (x) of disulfide-containing brancher in the hyperbranched core as reported in Table 1. Yellow circles illustrate -S-S- bonds and green circles illustrate -N3 functionality.


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favor the branching SCVP mechanism in relation to the formation of linear segments.59 The overall inimer amount in the polymerizations was retained at 20 mol %, while the BBEMA/BSSMA feed ratio was varied to change the amount of disulfide in the backbone of the hyperbranched polymers (Table 1). The inimer content was set to 20 mol % since it was hypothesized that this would result in a not to branched polymer structure, which would allow for enough space to facilitate guest molecule encapsulation. Furthermore, TBBPE (1), was used


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in the reaction since the use of a multifunctional initiator has shown to reduce the molecular weight dispersity in the SCVP reaction.60,

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The structural integrity of the hyperbranched

polymers was analyzed by 1H-NMR (ESI, Figure S11). Table 1. Molecular properties of the synthesized hyperbranched macroinitiators.

FS-S in entry

polymera

comonomer

brancher b

Mn, NMRc

Mn, SECd

(kDa)

(kDa)

Đd

(mol %) 1

HBMI(SS0)

HA

0

61

6.3

1.8

2

HBMI(SS45)

HA

45

52

10.3

1.5

3

HBMI(SS100)

HA

100

53

8.8

1.9

4

HBMI(SSN3)

AzSSMA

0

85

7.4

1.4

backbone -S-S-

pendant -S-S-

a

Hyperbranched copolymers of TBBPE:(BBEMA/BSSMA):comonomer, where x in (SSx) denotes molar fraction of disulfide-containing brancher determined from 1H NMR. b Molar fraction of disulfide-containing brancher determined from 1H NMR. c Assessed by 1H NMR. d Assessed by DMF-SEC calibrated with linear PMMA standards. The absence of visible vinyl signals in the spectra suggests that lower molecular weight species to a high extent have coupled to form hyperbranched polymers. This absence of residual vinyl bonds has earlier been reported for polymers synthesized to high monomer conversion via SCVP with relatively low inimer-to-comonomer ratio and a multifunctional initiating moiety.60 Furthermore, the methylene protons at 2.95 ppm in the 1H-NMR spectrum of HBMI(SS100) confirm the successful incorporation of disulfide bonds in the polymer backbone (ESI, Figure S11a). The HBMIs were further evaluated by size exclusion chromatography (SEC, RI) (Table 1 and ESI, Figure S12), showing monomodal traces with molecular weight dispersities of 1.5-1.9. The molecular weights are likely underestimated due to the lower hydrodynamic volume of branched macromolecules in relation to the linear PMMA standards used in SEC. Interference


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with the solvent front obstructed analysis of the HBMI by triple detection SEC. As discussed previously for similar polymers51, each HBMI is assumed to comprise one TBBPE moiety. By this assumption, molecular weights calculated from NMR indicate higher molecular weights compared to SEC. However, since NMR does not indicate connectivity caused by radical coupling, the true molecular weights of the hyperbranched polymers is most likely in between these two values. In addition, in the 1H-NMR spectra of the disulfide-containing polymer (HBMI(SS100)) a more pronounced peak at 1.97 ppm is seen compared to the polymer without inherent disulfide bonds (HBMI(SS0)) (ESI, Figure S11). This peak corresponds to methylenes adjacent to unreacted inimer initiating moieties57, 62, which suggest that the disulfide-containing hyperbranched polymers to a higher extent contain segments with a lower degree of branching. Table 2. Molecular properties of the synthesized hyperbranched dendritic-linear polymers.

entry

polymer

a

Mnb

Mwb

(kDa)

(kDa)

Đ

b

Dh,zc

D nd

c

PdI (nm)

(nm)

5

HBDLP(SS0)

450

800

1.9

39 ± 1

0.16

13 ± 4

6

HBDLP(SS45)

300

800

2.5

37 ± 1

0.18

14 ± 4

7

HBDLP(SS100)

400

950

2.3

35 ± 1

0.11

16 ± 4

8

HBDLP(SSN3)

350

500

1.6

25 ± 1

0.19

9±3

a

Hyperbranched dendritic-linear polymers (HBDLP) synthesized by chain-extension with OEGMA from the hyperbranched macroinitiators (Table 1, entry 1-4). b Assessed by triple detection DMF-SEC. c Assessed by DLS (z-average) in PBS. d Assessed by cryogenic TEM. As shown in previous work, the HBMIs can be utilized to polymerize hydrophilic polymers of OEGMA to form amphiphilic HBDLPs. Chain-extension of the HBMIs (Table 1, entries 1-3) by ATRP was conducted in highly diluted toluene solutions (70 wt%) for around 18 hours, and stopped at approximately 50 % conversion to avoid undesired coupling reactions. The purified


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amphiphilic polymers (Table 2, entries 5-7) were analyzed by 1H-NMR (ESI, Figure S13). Methylene groups, corresponding to the ethylene glycol segments, are visible at 3.50-3.65 and 4.06 ppm. Further, signals from the methylenes of HA in the hyperbranched macroinitiator can be seen at 1.30 and 1.60 ppm. Analysis by SEC (Figure 1a-c) shows that the amphiphilic HBDLPs are of high molecular weight (Mw = 800-950 kDa, RALLS detector). Substantially high molecular weight has shown important for such HBDLPs to stabilize as unimolecular micelles in aqueous solution.51

Figure 1. Triple detection size exclusion chromatograms, RI detector (solid) and RALLS detector (dashed) a) HBDLP(SS0) b) HBDLP(SS45) c) HBDLP(SS100) d) HBDLP(SSN3). Noticeably, the SEC traces (RI detector) of the HBDLPs extended from the macroinitiators with BSSMA (Figure 1b-c) show a shoulder to lower molecular weight, which is more pronounced


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for HBMI(SS45) with a combination of BBEMA and BSSMA. As discussed earlier, this indicates that the initiating moiety of BSSMA is less reactive than BBEMA, which results in a higher probability for the formation of more linear polymers during the SCVCP reaction. Evidently, this is not detectable by SEC of the HBMIs, however; is more pronounced for the HBDLPs where the difference in the number of initiating groups for linear and branched polymers will have increased impact. Reductively cleavable azide-functional HBDLP (Scheme 1b) One major advantage of the proposed HBDLP platform is that the synthesis through chainwise polymerization allows for a convenient designer’s freedom of the unimolecular micelles for multiple purposes. To illustrate the versatility of the platform, the second part of this work included an exchange of the hydrophobic comonomer HA for a highly functional comonomer that will enable selective delivery of covalently linked hydrophobic cargoes. The monomer 2((2-(methacryloyloxy)ethyl)disulfanyl) ethyl 6-azidohexanoate (AzSSMA, 4), displaying vinyl, disulfide, and azide functionalities, was synthesized adopting a similar strategy as for BSSMA starting from bis(2-hydroxyethyl) disulfide (ESI, Figure S7). The AzSSMA structure was confirmed by 1H-,

13

C-NMR , and FTIR spectroscopies (Figure 2b and ESI, Figure S9). In 1H-

NMR, signals from vinylic protons (5.58 and 6.11 ppm), as well as the methylene protons adjacent to the disulfide (2.89-2.97 ppm) and azide (2.31-2.35 ppm) confirm the successful synthesis. This can also be seen in the FTIR spectrum where peaks corresponding to the vinyl (1637 cm-1), and azide (2093 cm-1) moieties are clearly visible. The monomer, AzSSMA, was copolymerized with the inimer, BBEMA, adopting the same procedure as described for the HBMIs above (Scheme 1b). The resulting polymer, HBMI(SSN3) (Table 1, entry 4) was characterized by 1H-NMR (Figure 2a) and SEC analysis (ESI, Figure S12d), confirming the


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successful formation of an equal HBMI as when using HA as comonomer. FTIR analysis established that the azide functionalities were preserved during the polymerization (ESI, Figure S10). Consequently, a HBMI with high density of disulfide-linked azide functionalities was successfully synthesized, HBMI(SSN3). The end-groups of HBMI(SSN3) were extended by ATRP of OEGMA (Scheme 1b) to form a HBDLP (Table 2, entry 8). Triple detection SEC revealed that a high molecular weight polymer was formed, however; with somewhat lower average molecular weight compared to HBMIs with HA as comonomer. This may be a consequence of the more “bulky” pendant group of AzSSMA compared to HA. In the synthesis of the hyperbranched macroinitiator this may favor the chainwise mechanism over the step-wise in the SCVCP reaction, thus resulting in a higher number of concealed initiating sites, less available for chain extension.


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Figure 2. 1H-NMR (CDCl3, 400 MHz) of a) HBMI(SSN3) and b) AzSSMA monomer. Yellow and green circles symbolize -S-S- bonds and -N3 bonds, respectively. Formation of unimolecular micelles The amphiphilic core-shell type architecture of the HBDLPs, with a hydrophobic hyperbranched core surrounded by a high number of linear hydrophilic polymers is, as discussed earlier, a promising design to form stable NPs in aqueous solution. As we reported recently, at sufficiently high molecular weight and number of linear arms the polymers can stabilize individually, without any self-assembly, i.e. they form unimolecular micelles.51 There are several


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methods utilized to disperse amphiphilic polymers into macromolecular assemblies in aqueous media, including: direct dissolution, dialysis, nanoprecipitation, and methods involving slow evaporation of an organic solvent. Our synthesized HBDLPs have conveniently proven to form stable NPs by direct dissolution of the polymer in phosphate buffer saline (PBS) for 24 hours on a shaking device. Results from dynamic light scattering (DLS) of unfiltered PBS solutions (1.25 mg mL-1) of the HBDLPs are shown in Figure 3a (and ESI, Figure S19). The polymers form NPs with diameters of 35-39 nm, and interestingly low DLS polydispersity indices (PdI = 0.11-0.19) for such complex polymer architectures. Cryogenic TEM shows that the NPs formed are predominantly circular micellar structures, which are expected due to the polymer architecture and the high amount of hydrophilic components (Figure 3c and ESI, Figure S15). The smaller sizes observed by TEM compared to DLS is most likely because only the dense hydrophobic core provides enough contrast to be observed. To study the NP stability, the hydrodynamic diameter as a function of concentration was evaluated by DLS (Figure 3b and ESI, Figure S14). As seen in Figure 3b, the NPs formed from HBDLP(SS100) are hardly affected by a change in concentration from 0.1-10 mg mL-1, thus this hundred fold increase in concentration only lowered the NP size by a few nanometers (40 ± 3 nm to 33 ± 1 nm). This is more likely an effect of the difference in the surrounding media at 10 mg mL-1 than an indication of a size decrease caused by a rearrangement in a dynamic assembly.


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Figure 3. a) Size distributions from DLS of the HBDLPs in PBS (1.25 mg mL-1) b) Size distributions from DLS of HBDLP(SS100) at varying concentrations in PBS (0.1-10 mg mL-1) c) TEM of HBDLP(SS100).


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Consequently, the HBDLPs are believed to stabilize individually as unimolecular micelles. In addition, the sizes of the NPs show good thermal stability with no size difference between 25 ˚C and 37 ˚C. However, noticeably at lower NP concentrations (≤ 0.5 mg ml-1) the count rate is significantly lowered in DLS and some very small amount of 4000 nm objects occur (1-4 intensity %). This indicates that some very low amount of interparticle association is present, which is more pronounced at lower concentrations. Reductive biodegradation of the HBDLPs and their therapeutic delivery One objective of this work was to investigate if selective biodegradation in relevant concentrations of reducing agent could be enabled by introduction of disulfides to the backbone of the unimolecular carriers. Disulfide linkages have emerged as attractive tools to allow for selective intracellular response. The intracellular environment is highly reductive due to the presence of the low molecular weight thiol, Glutathione (GSH, 0.5-10 mM), while extracellular fluids are oxidative but still with low amounts of GSH (2-20 µM).63 As the synthesized HBDLPs are suggested to form unimolecular micelles it was evaluated if they could be selectively degraded in reductive environment. Dithiothreitol (DTT) is a commonly used reducing agent, known to effectively cleave disulfide bonds. The unimolecular micelles were treated with different concentrations of DTT (10 µM, 1 mM, and 10 mM) for 1 and 24 hours, and subsequently analyzed by SEC. Figure 4 show SEC traces of the HBDLPs (Table 2, entries 5-7) after treatment with DTT solution for 1 hour (no further change was seen after 24 h). The HBDLPs comprise similar amount of branching in their core, however; their branches contain different amount of BBEMA vs BSSMA, i.e. different number of the branches contain disulfide bonds. HBDLP(SS0), the control polymer, which do not contain any disulfide bonds, remain unaffected by the DTT treatment, thus confirming that DTT does not cause any unexpected


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degradation of the unimolecular micelles. Interestingly, the HBDLPs containing disulfide linkages in their core show a concentration-dependent reduction in molecular weight by DTT treatment, assigned to the reductive cleavage of their inherent disulfide bonds. After 1 hour treatment with 10 µM DTT (comparable to plasma concentration55, 64), the HBDLPs are nearly unaffected, indicating good stability. However, with 10 mM DTT (comparable to intracellular concentration56, 65), HBDLP(SS100) are degraded to 1/7 of its original molecular weight (Mp, 37 kg mol-1 with linear PMMA standards). Since no further degradation was seen after 24 hours it was concluded that full degradation was reached already after 1 hour. Results for HBDLP(SS45) with approximately 50 % of the branching units containing disulfide bonds showed similar behavior. Linear cleaved segments appear after 10 mM DTT treatment, and simultaneously the main HBDLP is somewhat degraded but remains quite intact due to the remaining “uncleavable” BBEMA segments. Conclusively, the disulfide-containing inimer BSSMA, enable introduction of degradable linkers to the backbone of the unimolecular micelles, and allow for tuning of the biodegradability of the polymers. Moreover, the clear difference in degradation between 10 µM and 10 mM DTT suggest that the NPs are suitable for selective degradation under intracellular conditions, but remain relatively stable during circulation. As previously discussed, the reductive environment in cells is retained by GSH. To further study the applicability of the polymers, the degradability of HBDLP(SS100) in GSH media was evaluated by SEC (ESI, Figure S16). As expected GSH is a less effective reducing agent compared to DTT. Treatment with 10 mM GSH (24 h) only resulted in partial degradation of the HBDLPs. This indicates that the polymers can be rapidly degraded by intracellular levels of DTT, but the non-quantitative reduction by GSH suggests that the disulfide linkers may need to be made more available for reduction to allow for effective biodegradation.


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Figure 4. Size exclusion chromatograms (RI detector) after treatment with different DTT concentrations a) HBDLP(SS0) b) HBDLP(SS45) c) HBDLP(SS100). As described, the synthesized unimolecular micelles could be degraded to different extents due to their varying degree of disulfide bonds in their hyperbranched backbones. It was further


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analyzed if this could be utilized to selectively tune the release of the chemotherapeutic doxorubicin (DOX), physically encapsulated in the core of the NPs. DOX was loaded to the NPs employing a procedure reported previously66, and the in vitro release was analyzed at different DTT concentrations (ESI, Figure S20). All polymers show diffusion-controlled release profiles and release 25-50 % of DOX within 72 hours. Surprisingly, no clear trend can be seen either when comparing the release between the different NPs, or between the release from each NP in pure PBS, 10 mM DTT and 100 mM DTT. Since degradation to varying extents was confirmed by SEC it was anticipated that this would be reflected in the release kinetics of DOX. One probable explanation to the low effect is that the polymers formed after degradation still are highly amphiphilic, thus resulting in a more or less stable assembly in aqueous solution. Thereby, the DOX release is still mainly controlled by diffusion as there is no continual circulation or convection within the inner dialysis membrane in this release model as the case in vivo would allow for.


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Figure 5. Cell viability of the breast cancer cell line MCF-7 after 48 hours of incubation with varying concentrations of a) neat HBDLP NPs b) DOX-loaded HBDLP(SS100) NPs, with and without pre-treatment of cells with 10 mM glutathione (GSH). To further study the delivery properties of DOX by the disulfide-containing carriers, cell viability of the common breast cancer cell line MCF-7 was analyzed by MTT assay. As seen in Figure 5a the neat HBDLPs are non-toxic to the cells up to 100 µg mL-1 when incubated for 48 hours. Furthermore, HBDLP(SS100) was chosen to study the delivery behavior of DOX, and showed a dose-dependent toxicity to the cells (Figure 5b). After 48 hours treatment the cell viability is reduced to 30 % as compared to 54 % for free DOX, which indicates that the NPs facilitate the delivery of DOX to the cells. However, when the MCF-7 cells are pretreated with the reducing agent GSH (5-20 mM), no additional effect is seen to the cell viability (Figure 5b and ESI Figure S21), which correlates well with results from the in vitro release test. This


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confirms that introduction of disulfide bonds in the backbone of the NPs may indeed be a promising way to allow for biodegradation of the high molecular weight HBDLPs, but to be a suitable approach for selective therapeutic delivery, the amphiphilic nature of the degradation products need to be decreased to avoid subsequent potential re-assembly. Disperse red conjugation and selective release One proposed strategy to avoid premature release of a therapeutic during circulation is covalent attachment to the polymer instead of physical entrapment in the hydrophobic core. This would ensure protection of the therapeutic and reduce the uncertainty of diffusion-controlled release during delivery. However, this simultaneously requires that the drug can be cleaved off at the site of action to ensure the therapeutic effect. As described earlier, HBDLP(SSN3) (Table 2, entry 8), with pendant disulfide-linked azide functionalities in the hyperbranched core was successfully synthesized. These interesting unimolecular NPs may have potential to selectively deliver a low molecular weight, hydrophobic cargo as a response to a reductive stimulus. To illustrate this, the hydrophobic dye, disperse red (DR), was chosen as a model compound. Acetylene-functional DR (DRA, 8) was synthesized and conjugated to the core of HBDLP(SSN3) by a copper(I)-catalyzed azide-alkyne cycloaddition in a THF/H2O solvent mixture. The pure product, HBDLP(SSDR), was achieved after extensive purification by precipitation and subsequent dialysis in ethanol, and the successful conjugation was confirmed by 1H-NMR and UV-Vis analyses. In the 1H-NMR spectra of HBDLP(SSDR), aromatic signals and the shifted methylene from DR, as well as the new proton in the triazole adduct, are visible (Figure 6c).


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Figure 6. 1H-NMR (CDCl3, 400 MHz) showing the disperse red (DR) conjugation to HBDLP(SSN3). a) HBDLP(SSN3) b) disperse red acetylene (DRA, 8) c) HBDLP(SSDR) after purification. Insets show PBS solutions of i) pure DRA ii) HBDLP(SSDR). The inset pictures in Figure 6 show the enhanced water solubility of DR when conjugated to the polymer. The successful conjugation was further confirmed by UV-Vis analysis by the appearance of the absorbance peak at 483 nm (ESI, Figure S17). The small shift between the free DR and HBDLP(SSDR) in the spectrum (494 to 483 nm in ethanol) indicates the change to a more hydrophobic environment after conjugation of DR to the NP core. The conjugation efficiency was determined to 61 % by UV-Vis, which means that in average 113 DR molecules are conjugated to each unimolecular micelle. As expected, the analysis of HBDLP(SSDR) by SEC and DLS shows that the conjugation resulted in an increase in hydrodynamic volume (ESI,


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Figures S18 and S19). Furthermore, after treatment with 10 mM DTT, the hydrodynamic volume of the original HBDLP(SSN3) was almost regained, thus demonstrating that the dye can be selectively cleaved off from the polymer (ESI, Figure S18). The selective delivery of the model dye, DR, from HBDLP(SSDR), was evaluated by a release test in PBS solutions. The test was performed in Slide-A-Lyzer@ MINI dialysis devices, and the dialysis media investigated were reductive solutions of DTT (10 µM and 10 mM), GSH (10 mM), and pure PBS as control. The results of the 72 hours release experiments are reported in Figure 7. In PBS, serving as control, the NPs initially (0-5 h) show no release of DR. However, after 24 hours about 7 % of DR is released from the NPs, but no additional release is detected after 72 hours. Since no reductive agent is present, this indicates that a small amount of unconjugated DR is still entrapped in the NPs despite the extensive purification efforts, and suggests that the unbound dye diffuses out with time. At 10 µM DTT, the proposed concentration in plasma, the disulfide bonds are fairly

Figure 7. Accumulative release of disperse red from HBDLP(SSDR) NPs in different aqueous reducing conditions. Insets show solutions of HBDLP(SSDR) (5 mg mL-1) after 72 h treatment with PBS (bottom) and 10 mM DTT (top). stable and only a low amount of dye is released. The initial small burst release at 10 µM indicates that the disulfide bonds at the hydrophobic-hydrophilic interface may be accessible for


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reduction, however; the subsequent stabilization of the release shows that most of the hydrophobic core is unaccessible at this low DTT concentration. Interestingly, when the DTT concentration is increased to the proposed intracellular concentration (10 mM), an instant and distinct release of the DR is observed (42 % in 5 h). Clearly, at this reducing concentration, a higher amount of the disulfide bonds are accessible and cleaved almost instantaneously. The DR release from the NPs at 10 mM GSH concentration initially show a significant difference compared to pure PBS, but is still much less effective in reducing the disulfide bonds than DTT, as discussed previously. This difference may to some extent be attributed to steric reasons, where the larger size of GSH compared DTT may obstruct the accessibility of the disulfide bonds. The results suggest that the synthesized unimolecular micelles allow for selective delivery as a response to a biological trigger. However, in similarity to the degradation studies with SEC, the less effective release by GSH indicates that the internal disulfide bonds might need to be more accessible to ensure a more pronounced response.

CONCLUSIONS We herein report a versatile route to redox-responsive unimolecular micelles through a twostep

synthesis

of

amphiphilic,

hyperbranched

dendritic-linear

polymers

(HBDLP).

Hyperbranched macroinitiators (HBMI) with disulfide linkages in the main chain, or in the pendant groups, were realized by SCVCP of functional starting materials. By subsequent chainextension with OEGMA, high molecular weight HBDLPs were achieved and proved to form individually stabilized unimolecular micelles in the size range 25-40 nm as determined by DLS. The unimolecular micelles with disulfide bonds in the hyperbranched backbone, showed up to a sevenfold molecular weight reduction under mimicked reductive intracellular conditions, and the


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biodegradability could be tailored by the amount of introduced disulfides. However, the difference in degree of degradation could not be employed to selectively tune the release behavior of the model cancer therapeutic Doxorubicin (DOX) from the unimolecular micelles. The retained amphiphilicity of the polymers after degradation is suggested to initiate selfassembly of the degradation products, and no trend could be observed for the triggered DOX release behavior from the different HBDLPs under reductive environment. In addition, highly functional unimolecular micelles, displaying disulfide-linked azide moieties in their hyperbranched core, were produced by utilizing the novel, multifunctional, monomer, 2-((2(methacryloyloxy)ethyl)disulfanyl) ethyl 6-azidohexanoate (AzSSMA). The resulting, stable, unimolecular micelles allowed for post-functionalization with the hydrophobic model dye, disperse red, and were proved to instantly release the dye molecules under reductive conditions. This selectively triggered release characteristic, in combination with the potential biodegradability of the high molecular weight unimolecular micelles, proposes a versatile platform for controlled drug delivery.

ASSOCIATED CONTENT Supporting Information Available Additional synthetic protocols, 1H and

13

C NMR, FT-IR, SEC, DLS, TEM, and cell viability

results. This material is available at free of charge via the Internet at http://pubs.acs.org

AUTHOR INFORMATION Corresponding author


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* [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources Swedish Research Council (grants 2011-4995 and 2009-3259)

ACKNOWLEDGMENT Funding support by the Swedish Research Council (VR), grants 2011-4995 and 2009-3259, are greatly acknowledged. Dr. Pierre Chambon is greatly thanked for help with triple detection SEC. Prof. Olli Ikkala is acknowledged for fruitful discussion and support.

REFERENCES (1) Matyjaszewski, K. Macromolecules 2012, 45 (10), 4015-4039 (2) Matyjaszewski, K.; Tsarevsky, N.V. J. Am. Chem. Soc. 2014, 136 (18), 6513-6533 (3) Moad, G.; Rizzardo, E.; Thang, S.H. Polymer 2008, 49 (5), 1079-1131 (4) Rizzardo, E.; Solomon, D.H. Aust. J. Chem. 2012, 65 (8), 945-969 (5) Blanazs, A.; Armes, S.P.; Ryan, A.J. Macromol. Rapid Commun. 2009, 30 (4-5), 267-277 (6) Discher, D.E.; Eisenberg, A. Science 2002, 297 (5583), 967-973


36

Page 37 of 50

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

Biomacromolecules

(7) Duncan, R. Nat. Rev. Drug Discovery 2003, 2 (5), 347-360 (8) Kataoka, K.; Harada, A.; Nagasaki, Y. Adv. Drug Delivery Rev. 2001, 47 (1), 113-131 (9) Miyata, K.; Christie, R.J.; Kataoka, K. React. Funct. Polym. 2011, 71 (3), 227-234 (10) Wang, Y.; Grayson, S.M. Adv. Drug Delivery Rev. 2012, 64 (9), 852-865 (11) Elsabahy, M.; Wooley, K.L. Chem. Soc. Rev. 2012, 41 (7), 2545-2561 (12) Ferrari, M. Nat. Rev. Cancer 2005, 5 (3), 161-171 (13) Owens, D.E., 3rd; Peppas, N.A. Int. J. Pharm. 2006, 307 (1), 93-102 (14) Fox, M.E.; Szoka, F.C.; Fréchet, J.M.J. Acc. Chem. Res. 2009, 42 (8), 1141-1151 (15) Schroeder, A.; Heller, D.A.; Winslow, M.M.; Dahlman, J.E.; Pratt, G.W.; Langer, R.; Jacks, T.; Anderson, D.G. Nat. Rev. Cancer 2012, 12 (1), 39-50 (16) Matsumura, Y.; Maeda, H. Cancer Res. 1986, 46 (12, Pt. 1), 6387-6392 (17) Maeda, H.; Wu, J.; Sawa, T.; Matsumura, Y.; Hori, K. J. Controlled Release 2000, 65 (12), 271-284 (18) Torchilin, V.P. J. Controlled Release 2001, 73 (2-3), 137-172 (19) Chen, H.; Kim, S.; He, W.; Wang, H.; Low, P.S.; Park, K.; Cheng, J.-X. Langmuir 2008, 24 (10), 5213-5217 (20) Poon, Z.; Lee, J.A.; Huang, S.; Prevost, R.J.; Hammond, P.T. Nanomedicine (Philadelphia, PA, U. S.) 2011, 7 (2), 201-209


37

Biomacromolecules

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 38 of 50

(21) Newkome, G.R.; Yao, Z.; Baker, G.R.; Gupta, V.K. J. Org. Chem. 1985, 50 (11), 20032004 (22) O'Reilly, R.K.; Hawker, C.J.; Wooley, K.L. Chem. Soc. Rev. 2006, 35 (11), 1068-1083 (23) Read, E.S.; Armes, S.P. Chem. Commun. (Cambridge, U. K.) 2007, (29), 3021-3035 (24) Stenzel, M.H. Macromol. Rapid Commun. 2009, 30 (19), 1603-1624 (25) Elsabahy, M.; Wooley, K.L. J. Polym. Sci., Part A: Polym. Chem. 2012, 50 (10), 18691880 (26) Thurmond, K.B., II; Kowalewski, T.; Wooley, K.L. J. Am. Chem. Soc. 1996, 118 (30), 7239-7240 (27) Nyström, A.M.; Wooley, K.L. Acc. Chem. Res. 2011, 44 (10), 969-978 (28) Smeets, N.M.B. Eur. Polym. J. 2013, 49 (9), 2528-2544 (29) O'Brien, N.; McKee, A.; Sherrington, D.C.; Slark, A.T.; Titterton, A. Polymer 2000, 41 (15), 6027-6031 (30) He, T.; Adams, D.J.; Butler, M.F.; Cooper, A.I.; Rannard, S.P. J. Am. Chem. Soc. 2009, 131 (4), 1495-1501 (31) Weaver, J.V.M.; Williams, R.T.; Royles, B.J.L.; Findlay, P.H.; Cooper, A.I.; Rannard, S.P. Soft Matter 2008, 4 (5), 985-992 (32) He, T.; Adams, D.J.; Butler, M.F.; Yeoh, C.T.; Cooper, A.I.; Rannard, S.P. Angew. Chem., Int. Ed. 2007, 46 (48), 9243-9247


38

Page 39 of 50

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

Biomacromolecules

(33) Wang, K.; Peng, H.; Thurecht, K.J.; Puttick, S.; Whittaker, A.K. Polym. Chem. 2013, 4 (16), 4480 (34) Warren, N.J.; Armes, S.P. J. Am. Chem. Soc. 2014, 136 (29), 10174-10185 (35) Karagoz, B.; Esser, L.; Duong, H.T.; Basuki, J.S.; Boyer, C.; Davis, T.P. Polym. Chem. 2014, 5 (2), 350-355 (36) Moorefield, C.N.; Newkome, G.R. C. R. Chim. 2003, 6 (8-10), 715-724 (37) Gillies, E.; Fréchet, J. Drug Discovery Today 2005, 10 (1), 35-43 (38) Gitsov, I. J. Polym. Sci., Part A: Polym. Chem. 2008, 46 (16), 5295-5314 (39) Menjoge, A.R.; Kannan, R.M.; Tomalia, D.A. Drug Discovery Today 2010, 15 (5/6), 171185 (40) Voit, B.I.; Lederer, A. Chem. Rev. (Washington, DC, U. S.) 2009, 109 (11), 5924-5973 (41) Wurm, F.; Frey, H. Prog. Polym. Sci. 2011, 36 (1), 1-52 (42) Voit, B. J. Polym. Sci., Part A: Polym. Chem. 2000, 38 (14), 2505-2525 (43) Chen, G.; Wang, L.; Cordie, T.; Vokoun, C.; Eliceiri, K.W.; Gong, S. Biomaterials 2015, 47, 41-50 (44) Aryal, S.; Prabaharan, M.; Pilla, S.; Gong, S. Int. J. Biol. Macromol. 2009, 44 (4), 346352 (45) Xu, S.; Luo, Y.; Graeser, R.; Warnecke, A.; Kratz, F.; Hauff, P.; Licha, K.; Haag, R. Bioorg. Med. Chem. Lett. 2009, 19 (3), 1030-1034


39

Biomacromolecules

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 40 of 50

(46) Li, X.; Qian, Y.; Liu, T.; Hu, X.; Zhang, G.; You, Y.; Liu, S. Biomaterials 2011, 32 (27), 6595-6605 (47) Zeng, X.; Zhang, Y.; Wu, Z.; Lundberg, P.; Malkoch, M.; Nyström, A.M. J. Polym. Sci., Part A Polym. Chem. 2012, 50 (2), 280-288 (48) Yang, X.; Grailer, J.J.; Pilla, S.; Steeber, D.A.; Gong, S. Bioconjugate Chem. 2010, 21 (3), 496-504 (49) Fréchet, J.M.J.; Henmi, M.; Gitsov, I.; Aoshima, S.; Leduc, M.R.; Grubbs, R.B. Science (Washington, D. C.) 1995, 269 (5227), 1080-1083 (50) Min, K.; Gao, H. J. Am. Chem. Soc. 2012, 134 (38), 15680-15683 (51) Porsch, C.; Zhang, Y.N.; Ducani, C.; Vilaplana, F.; Nordstierna, L.; Nyström, A.M.; Malmström, E. Biomacromolecules 2014, 15 (6), 2235-2245 (52) Bektas, S.; Ciftci, M.; Yagci, Y. Macromolecules (Washington, DC, U. S.) 2013, 46 (17), 6751-6757 (53) Du, W.; Nyström, A.M.; Zhang, L.; Powell, K.T.; Li, Y.; Cheng, C.; Wickline, S.A.; Wooley, K.L. Biomacromolecules 2008, 9 (10), 2826-2833 (54) Markovsky, E.; Baabur-Cohen, H.; Eldar-Boock, A.; Omer, L.; Tiram, G.; Ferber, S.; Ofek, P.; Polyak, D.; Scomparin, A.; Satchi-Fainaro, R. J. Controlled Release 2012, 161 (2), 446-460 (55) Saito, G.; Swanson, J.A.; Lee, K.-D. Adv. Drug Delivery Rev. 2003, 55 (2), 199-215 (56) Fleige, E.; Quadir, M.A.; Haag, R. Adv. Drug Delivery Rev. 2012, 64 (9), 866-884


40

Page 41 of 50

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Biomacromolecules

(57) Tsarevsky, N.V.; Huang, J.; Matyjaszewski, K. J. Polym. Sci., Part A: Polym. Chem. 2009, 47 (24), 6839-6851 (58) Graff, R.W.; Wang, X.; Gao, H. Macromolecules (Washington, DC, U. S.) 2015, 48 (7), 2118-2126 (59) Chen, Z.-C.; Chiu, C.-L.; Huang, C.-F. Polymers (Basel, Switz.) 2014, 6 (10), 2552-2572, 2521 pp. (60) Hong, C.-Y.; Zou, Y.-F.; Pan, C.-Y. Polym. Int. 2003, 52 (2), 257-264 (61) Zhou, Z.P.; Yan, D.Y. Sci. China: Chem. 2010, 53 (12), 2429-2439 (62) Cai, Y.; Liu, Y.-Y. Macromol. Chem. Phys. 2013, 214 (8), 882-891 (63) Meng, F.; Hennink, W.E.; Zhong, Z. Biomaterials 2009, 30 (12), 2180-2198 (64) Jones, D.P.; Carlson, J.L.; Samiec, P.S.; Sternberg, P., Jr.; Mody, V.C., Jr.; Reed, R.L.; Brown, L.A.S. Clin. Chim. Acta 1998, 275 (2), 175-184 (65) Hassan, S.S.M.; Rechnitz, G.A. Anal. Chem. 1982, 54 (12), 1972-1976 (66) Porsch, C.; Zhang, Y.; Östlund, Å.; Damberg, P.; Ducani, C.; Malmström, E.; Nyström, A.M. Part. Part. Syst. Charact. 2013, 30 (4), 381-390


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Disulfide-Functionalized Unimolecular Micelles as Selective Redox

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