Tuning amphiphilicity/temperature-induced self-assembly and in-situ disulfide crosslinking of reduction-responsive block copolymers

Tuning Amphiphilicity/Temperature-Induced Self-Assembly and In-Situ

Disulfide Crosslinking of Reduction-Responsive Block Copolymers

Nicky Chan, Nathan Yee, So Young An, Jung Kwon Oh

Department of Chemistry and Biochemistry and Center for Nanoscience Research (CENR), Concordia University, Montreal,

Quebec, Canada H4B 1R6

Correspondence to: J. K. Oh (E-mail: [email protected])

Received 21 March 2014; accepted 16 April 2014; published online 29 April 2014

DOI: 10.1002/pola.27216

ABSTRACT: New poly(ethylene oxide)-based block copolymers

(ssBCs) with a random copolymer block consisting of a

reduction-responsive disulfide-labeled methacrylate (HMssEt)

and a thermoresponsive di(ethylene glycol)-containing methac-

rylate (MEO2MA) units were synthesized. The ratio of HMssEt/

MEO2MA units in the random P(MEO2MA-co-HMssEt) copoly-

mer block enables the characteristics of well-defined ssBCs to

be amphiphilic or thermoresponsive and double hydrophilic.

Their amphiphilicity or temperature-induced self-assembly

results in nanoaggregates with hydrophobic cores having dif-

ferent densities of pendant disulfide linkages. The effect of

disulfide crosslinking density on morphological variation of

disulfide-crosslinked nanogels is investigated. In response to

reductive reactions, the partial cleavage of pendant disulfide

linkages in the hydrophobic cores converts the physically asso-

ciated aggregates to disulfide-crosslinked nanogels. The occur-

rence of in-situ disulfide crosslinks provides colloidal stability

upon dilution. VC 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part

A: Polym. Chem. 2014, 52, 2057–2067

KEYWORDS: atom transfer radical polymerization (ATRP); block

copolymers; self-assembly; stimuli-sensitive polymers

INTRODUCTION Well-defined block copolymers have beenextensively used as effective building blocks in the construc-tion of multifunctional and nanostructured materials used astemplates, coatings, elastomers, and nanomedicine.1–8 Theirversatility and diverse applications stem from their ability toundergo thermodynamically driven microscopic phase-separation and self-assembly enabling the fabrication ofnovel nanomaterials with interesting morphologies. A prom-ising class of nanomaterials based on block copolymers isself-assembled nanometer-sized aggregates in aqueous solu-tion.9–12 One type of the block copolymers is amphiphilicblock copolymers (ABPs) that consist of hydrophobic andhydrophilic blocks. Through amphiphilicity-driven self-assembly, ABPs form micellar aggregates containing hydro-phobic cores surrounded with a hydrophilic corona. Anotherexample is stimuli-responsive double hydrophilic blockcopolymers (DHBCs), particularly thermoresponsive DHBCs.They consist of a hydrophilic block and a thermoresponsiveblock which responds to temperature change.13–19 At condi-tions above the lower critical solution temperature (LCST) ofthe thermoresponsive block, DHBCs undergoes a coil to glob-ular transition to form self-assembled aggregates throughtemperature-driven self-assembly. For both ABP-based andDHBC-based self-assembled aggregates, their inner cores

enable the encapsulation of hydrophobic therapeutics; thus,they are considered as promising nanocarriers in pharma-ceutical science and nanomedicine.

However, colloidal stability of physical aggregates upon dilu-tion remains a challenge. A large dilution upon intravenousinjections presents the aggregates with local environmentsfar below the critical aggregation concentration. Conse-quently, the dilution leads to destabilization or dissociationof the aggregates, which in turn leads to premature releaseof encapsulated drugs. A proposed strategy to improve thestability issue has been to develop crosslinked micelles (i.e.nanogels).20–22 However, the use of the permanent covalentcrosslinks hampers the controlled release of encapsulateddrugs. One potential solution that has been explored is tocreate new labile crosslinks, typically disulfide linkages, toyield core-crosslinked nanogels through in situ formation ofnew disulfide crosslinks.23 Different from an introduction ofdisulfides through the use of disulfide-labeled cross-linkers,24–31 the in situ formation of disulfide linkagesinvolves thiol-disulfide polyexchange reactions of pendantdisulfides of polymer chains in the presence of catalyticamounts of thiol reducing agents. This strategy generatesnanogels crosslinked with new labile disulfide crosslinks,

Additional Supporting Information may be found in the online version of this article.

VC 2014 Wiley Periodicals, Inc.

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affording enhanced colloidal stability. Later, the newly-formed disulfide crosslinks can be cleaved in the presence ofexcess reducing agents, causing disulfide-crosslinked nano-gels to dissociate, and thus enhancing the release of encap-sulated drugs in targeted cells.32,33 Several reports describereduction-responsive nanogels based on homopolymers suchas poly(amidoamine),34 dextrans,35,36 and random copoly-mers,37–40 as well as block copolymers.41,42 Our group hasalso reported the synthesis and characterization of well-defined diblock copolymers consisting of a hydrophilic poly(-ethylene oxide) (PEO) block and a hydrophobic polymetha-crylate containing pendant disulfide linkages (HMssEt).Preliminary investigations of the self-assembly of this ABPinto micelles and reduction-responsive release as a functionof morphology change was also conducted.43 Despite theseadvances, further understanding of the in situ disulfide cross-linking strategy, in particular effects of crosslinking densityand reducing agent concentration is required for effectivetherapeutic applications of disulfide based nanogels.

In this work, we investigated the effect of disulfide crosslink-ing density and reducing agent concentration on morphologi-cal variation of disulfide-crosslinked nanogels (Scheme 1).Atom transfer radical polymerization (ATRP)44 was used tosynthesize a series of new block copolymers consisting of aPEO block and a random copolymer block consisting of boththermoresponsive di(ethylene glycol) monomethyl ethermethacrylate (MEO2MA) and reduction-responsive HMssEt,thus yielding PEO-b-P(MEO2MA-co-HMssEt) copolymers.These ABPs with pendant disulfide linkages (ssBCs) aredesigned to have varying ratios of HMssEt/MEO2MA units inthe random copolymer block, creating self-assembled aggre-gates with consistent hydrophobic core size and differentdensities of pendant disulfide linkages. Further, dependingon the amount of hydrophobic HMssEt units in the copoly-mers, these ssBCs can be amphiphilic or thermoresponsiveand double hydrophilic, thus leading to self-assembly drivenby amphiphilicity or temperature change. Later, the partialcleavage of pendant disulfide linkages in the hydrophobiccores resulted in the conversion of physically associated

micelles to in situ disulfide-crosslinked nanogels. The occur-rence of new disulfide crosslinks provides colloidal stabilityupon dilution.

EXPERIMENTAL

Instrumentation and Analyses1H-NMR spectra were recorded using a 500 MHz Varianspectrometer. The CDCl3 singlet at 7.26 ppm was selected asthe reference standard. Monomer conversion was deter-mined using 1H-NMR for aliquots taken during polymeriza-tion. Molecular weight and molecular weight distributionwere determined by gel permeation chromatography (GPC).An Agilent GPC was equipped with a 1260 Infinity IsocraticPump and a RI detector. Two Agilent PLgel mixed-C andmixed-D columns were used with DMF containing 0.1 mol %LiBr at 50 �C at a flow rate of 1.0 mL/min. Linear poly(methyl methacrylate) standards from Fluka were used forcalibration. Aliquots of polymer samples were dissolved inDMF/LiBr. The clear solutions were filtered using a 0.25 mmPTFE filter to remove any solvent-insoluble species. A dropof anisole was added as a flow rate marker.

Dynamic Light ScatteringThe size and size distribution of ssBC-based aggregates andtheir crosslinked nanogels in hydrodynamic diameter byvolume was measured at a fixed scattering angle of 175� at25 �C with a Malvern Instruments Nano S ZEN1600equipped with a 633 nm He-Ne gas laser.

MaterialsCopper(II) bromide (CuBr2, >99.99%), tin(II) 2-ethylhexanoate(Sn(EH)2, 95%), and nile red (NR) from Aldrich, and DL-dithiothreitol (DTT, 99%) from Acros Organics were purchasedand used as a received. Di(ethylene glycol) methyl ether methac-rylate (MEO2MA, 95%) were purchased from Aldrich and puri-fied by passing through a column filled with basic alumina toremove inhibitors before used. Tris(2-pyridylmethyl)amine(TPMA),45 PEO-functionalized 2-bromoisobutyrate (PEO-Br),46

and HMssEt (a methacrylate bearing a pendant disulfide

SCHEME 1 Aqueous self-assembly and in situ disulfide crosslinking of ssBCs with various ratios of HMssEt/MEO2MA units. [Color

figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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linkage)43 were synthesized as described according to literatureprocedures.

Synthesis of ssBCs Using ARGET ATRPA series of ssBC diblock copolymers were synthesized byARGET ATRP of mixtures consisting of MEO2MA and differ-ent amounts of HMssEt in anisole at 40 �C mediated with aCuBr2/TPMA complex in the presence of PEO-Br macroinitia-tor. As a typical example to synthesize ssBC-10 containing10 mol % HMssEt in feed, PEO-Br (0.39 g, 76 mmol),MEO2MA (0.77 g, 4.1 mmol), HMssEt (0.16 g, 0.46 mmol),TPMA (3.3 mg, 11.4 mmol), CuBr2 (0.85 mg, 3.8 mmol), andanisole (3.5 g) were mixed in a 10 mL Schlenk flask. Themixture was deoxygenated by purging under nitrogen for 1h and placed in an oil bath at 40 �C. A nitrogen-prepurgedsolution of Sn(EH)2 (12.3 mg, 30 mmol) dissolved in anisole(0.5 g) was injected into the Schlenk flask to initiate poly-merization. Polymerization was stopped by cooling the reac-tion vessel and exposing the contents to air.

For purification, the as-synthesized polymer solutions werediluted with acetone and then passed through a basic alu-mina column to remove residual copper. Acetone wasremoved by rotary evaporation at room temperature. Theresidues were precipitated from cold hexane three times andthen dried under vacuum at room temperature for 18 h.

Determination of LCST of Thermoresponsive ssBCsLight scattering (LS) intensity (count rates) of aqueous copoly-mer solutions was measured using DLS. For the measure-ments, aliquots of the dried ssBC (30 mg) were dissolved indeionized water (10 mL) to form clear aqueous solutions at 3mg/mL. The solutions were then filtered through 0.45 lm PESfilters to remove large aggregates. The temperature was variedfrom 5 to 85 �C by an increment of 1 �C, and three measure-ments were made with a total of six scans at each tempera-ture. LCST was determined from the onset of increase in LSintensity.

Aqueous Assembly and Disassembly ofThermoresponsive ssBCsAn aliquot of the dried ssBC (10 mg) was dissolved in deion-ized water (10 mL) to form a clear aqueous solution at 1mg/mL concentration. The resulting solution was then fil-tered through a 0.45 mm PES filter to remove large aggre-gates. The temperature was periodically varied between 1 �C(far below LCST) and 50 �C (far above LCST) to measuresize distribution using DLS, with an equilibration time of 25min between each measurement.

Determination of Critical Micellar Concentration ofAmphiphilic ssBCsA stock solution of NR in THF at 1 mg/mL and stock solutionsof ssBCs in THF at 1 mg/mL and 5 mg/mL were prepared.Then, different amounts of the ssBC stock solutions weremixed with the same amount of the NR stock solution (0.5mL) in THF to prepare polymer and NR mixtures at increasingssBC concentrations. Additional THF was then added to theresulting ssBC and NR mixture such that the total volume of

the final polymer and NR THF solution was adjusted to be 2mL. Water (10 mL) was then added drop-wise to the ssBCand NR mixtures, and then the resulting dispersions werestirred for 24 h to remove THF via evaporation. They werethen subjected to filtration using 0.45 mm PES filters toremove excess NR, yielding a series of NR-loaded micelles withvarious concentrations of ssBC ranging from 1026 to 0.1 mg/mL. Their fluorescence spectra were recorded with kex5 480nm to monitor the fluorescence intensity at kem5 620 nm.

Preparation of Aqueous Self-Assembled Micelles ofAmphiphilic ssBCsTo clear organic solutions consisting of the purified, driedssBC (15 mg) dissolved in THF (2 mL) in a 20 mL vial, water(15 mL) was added drop-wise using a syringe pump equippedwith a plastic syringe (20 mL volume and 20 mm diameter)at an addition rate of 0.5 mL/min. The resulting dispersionswere kept under stirring for 24 h at room temperature toremove residual THF. They were then filtered using a 0.25 mmPES filter to remove unexpected aggregates. Gas chromatogra-phy was used to confirm no residual THF remained in theresulting micellar dispersions at 1 mg/mL concentration.

In Situ Disulfide-Crosslinking and CharacterizationFor amphiphilic micelles, an aqueous micellar dispersion at 1mg/mL (4 mL) was mixed with different volumes of anaqueous stock DTT solution (5 mg/mL) using a syringepump with a plastic syringe (1 mL volume and 4.8 mmdiameter) at an addition rate of 0.1 mL/h. The amount ofDTT was calculated based on mole equivalent to disulfidelinkages in the micelles. The resulting mixture was stirred atroom temperature for 3 days to form colloidally stable cross-linked nanogel dispersions. To examine the occurrence ofsufficient crosslinking, aliquots (0.2 mL) of stable nanogelswere mixed with DMF and stirred for the given times. Themixture was then analyzed using DLS to track any changesin particle size due to swelling or polymer dissociation. Fortemperature-driven thermoresponsive aggregates, an aliquotof ssBC-10 (10 mg) was dissolved in deionized water (10mL). Different amounts of the aqueous DTT stock solution (5mg/mL) was added to the clear aqueous polymer solution.The resulting mixture was then placed into a heated oil bathset at 30 �C while stirring at 200 rpm for 3 days to form col-loidally stable core-crosslinked nanogels with in situ disul-fide bonds. To confirm the occurrence of core-crosslinking,the nanogels were cooled to 1 �C, well below the LCST tran-sition temperature of ssBC-10 for 30 min to trigger dissocia-tion of the polymer aggregates. Further, aliquots (0.2 mL) ofstable nanogels were mixed with DMF and stirred for thegiven times. Similarly, the mixture was then analyzed usingDLS.

RESULTS AND DISCUSSION

Synthesis of ssBCs Using ARGET ATRPAs illustrated in Scheme 2, well-defined ssBCs having differ-ent amounts of pendant disulfide linkages were synthesizedusing ATRP through an activators regenerated by electron



transfer process (ARGET ATRP).47,48 In the presence of aPEO-Br macroinitiator, a series of ARGET ATRP mediated bya CuBr2/TPMA complex was performed with mixtures ofMEO2MA and HMssEt (a methacrylate possessing a pendantdisulfide linkage) in anisole at 40�C. The amounts of HMssEtwere varied from 0 to 100 mol % in the feed. Their experi-mental conditions and final properties are summarized inTable 1.

For all five polymerizations with different amounts ofHMssEt, monomer conversion determined by 1H-NMRreached 60–65% after 4 h. All the resulting ssBC copoly-merization was well-controlled, with similar number-average molecular weight (Mn)5 17–21 kg/mol and narrowmolecular weight distribution as indicated by a low polydis-persity index (Mw/Mn)< 1.15. As seen in Supporting Infor-mation Figure S1, the GPC traces of all the blockcopolymers were shifted to high molecular weight region,suggesting successful chain extension from PEO-Br macroi-nitiator. Note that a small shoulder in low molecular weightregion is attributed to the presence of unreacted PEO-Br orunfunctionalized PEO, and thus has negligible impact on theself-assembly behavior. Figure 1 show the typical examplesof 1H-NMR spectra of ssBC-0 designed with no HMssEt andssBC-25 with 25 mol % HMssEt in CDCl3. Typical peaksinclude the peak at 3.4 ppm (d) corresponding to pendantmethoxy protons in MEO2MA units, the peak at 3.37 ppm

(e) to methoxy protons in PEO block, and the peak at 2.9ppm (f,g) to methylene protons adjacent to disulfide linkagein HMssEt units. From their integral ratios, the number ofMEO2MA and HMssEt units in the resulting ssBCs (DP) wascalculated as denoted in Table 1. These results suggest thatchain extension of PEO-Br with MEO2MA and HMssEt pro-ceeded in a living fashion, yielding well-defined ssBCcopolymers with different densities of pendant disulfidelinkages.

Evaluation of Thermoresponsive Properties of ssBCsUsing DLSThe resulting ssBCs consist of a hydrophilic PEO block anda thermoresponsive, reduction-responsive P(MEO2MA-co-HMssEt) random copolymer block with different amountsof MEO2MA units. DLS was used to examine the thermalproperties of the ssBCs. LS intensity of aqueous copolymersolutions at 3 mg/mL (0.3 wt %) was measured with tem-peratures varying from 5 to 85�C in increments of 1�C. Fig-ure 2 shows the evolution of normalized LS of twocopolymers ssBC-0 and ssBC-10 over this temperaturerange. Normalized LS intensity increased sharply with anincrease in temperature, suggesting the formation of largeparticles created by aggregation of polymer chains uponheating. Such a change is attributed to an increase in thehydrophobicity of PMEO2MA as temperature increased.The temperature at which the LS intensity increases

SCHEME 2 Synthesis of well-defined PEO-b-P(MEO2MA-co-HMssEt) block copolymers via ARGET ATRP.

TABLE 1 Experimental Conditions and Properties of ssBCs Having Different Densities of Pendant Disulfide Linkagesa

Copolymers

HMssEt in

feed (mol %) Convb Mnc (g/mol) Mw/Mn

c

DP in P(MEO2MA-HMssEt) blockd

HMssEt MEO2MA Total

ssBC-100 100 0.66 20,700 1.14 49 0 49

ssBC-50 50 0.61 20,600 1.11 24 25 49

ssBC-25 25 0.67 18,800 1.14 11 33 44

ssBC-10 10 0.57 18,000 1.11 5 37 42

ssBC-0 0 0.57 17,490 1.12 0 42 42

a Conditions: ([MEO2MA]0 1 [HMssEt]0)/[PEO-Br]0/[CuBr2]0/[TPMA]0/[Sn(EH)2]05 60/1/0.05/0.15/0.4, monomers/anisole5 0.25/1 wt/wt.b Determined by 1H-NMR.

c Determined by GPC calibrated with PMMA standards.d Determined by 1H-NMR.


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corresponds to critical micellar temperature (or cloudpoint) of the polymer in solution, and can be used as anapproximation of the LCST at a specific polymer concentra-

tion in solution.17,19,30 The LCST of the copolymers deter-mined from the onset of the increase in LS intensity was33�C for ssBC-0 containing 0 mol% HMssEt (e.g. PEO-b-PMEO2MA), whereas it was 23�C for ssBC-10 containing 10mol% HMssEt, which is lower by 10�C than that of ssBC-0.The lower cloud point is attributed to the increase inhydrophobicity of the copolymer by introducing hydropho-bic HMssEt units. However, a further increase in HMssEt to25 mol % (ssBC-25) does not exhibit well-defined LCSTbehavior (Supporting Information Figure S2). These resultssuggest ssBCs having HMssEt greater than 25 mol % areamphiphilic.

Temperature-Induced Assembly and Disassembly ofThermoresponsive ssBCsThe ssBC-10 having 10 mol % HMssEt units exhibits anLCST transition close to body temperature. Its temperature-driven self-assembly and disassembly in aqueous solution(pH5 6.5) was followed via DLS measurements. As seen inFigure 3(a), the diameter was determined to be �7 nm at1�C, below the LCST, which is attributed to the existence ofthe block copolymer as individual copolymer chains with arandom coil configuration. When the temperature was

FIGURE 1 Typical examples of 1H-NMR spectra of ssBC-0 designed with no HMssEt and ssBC-25 with 25 mol % HMssEt in CDCl3.

X denotes residual solvents or impurities.

FIGURE 2 Temperature dependence of normalized light scat-

tering intensity by DLS for 3 mg/mL aqueous solutions of

ssBC-0 and ssBC-10.



increased to 50�C, above the LCST, the diameter alsoincreased to �26 nm due to the formation of physical aggre-gates through self-assembly. Upon cooling to 1�C, the result-ing self-assembled aggregates were disassembled toindividual chains; subsequently, these chains again self-assembled to form aggregates with the diameter �26 nmupon heating to 50�C. As seen in Figure 3(b), the ssBC-10exhibit reversible self-assembly and disassembly behaviorupon subsequent heating and cooling processes.

In PBS (pH5 7), the diameter was determined to be �8 nmat 1�C and �26 nm at 50�C (Supporting Information FigureS3); these sizes are similar to those measured in water(pH5 6) above. These results suggest no significant effect ofionic strength on micelle size due to the presence of neutralPEO coronas.

Aqueous Self-Assembly of Amphiphilic ssBCsThree ssBC-100, ssBC-50, and ssBC-25 are amphiphilic;thus, their CMC was first determined using fluorescencespectroscopy with a Nile Red (NR) probe.49,50 A series ofmixtures consisting of the same amount of NR and variousamounts of ssBCs ranging from 1026 to 0.1 mg/mL in aque-ous solution were prepared. After removal of THF by sol-vent evaporation and free NR by filtration, theirfluorescence spectra were measured (Supporting Informa-tion Figure S4 [left]). The evolution of fluorescence inten-sity at the maximum wavelength over increasingconcentrations of ssBCs is plotted Supporting InformationFigure S4 (right). The fluorescence intensity was low anddid not change significantly at lower polymer concentra-tions since micelles were not formed to encapsulate NR.However, fluorescence rapidly increased with an increasingconcentration of ssBCs above the CMC as NR becameentrapped in a growing number of self-assembled micelles.Regression analysis of the two linear regions of the data

was used to determine the CMC to be 9 mg/mL for ssBC-100, 9 mg/mL for ssBC-50, and 8 mg/mL for ssBC-25. Theseresults suggest the formation of self-assembled micelles ofssBCs at similar concentrations. Note that the CMC forssBC-100 determined by a NR probe is smaller than that(49 mg/mL) determined by tensiometry.43 A similar compar-ison has been reported for other ABPs.51

At concentrations above the CMC, the three ssBCs self-assembled to form micellar aggregates consisting of a hydro-phobic P(MEO2MA-co-HMssEt) core and hydrophilic PEOcorona in aqueous solution. Figure 4 shows the DLS dia-grams of the self-assembled aggregates in aqueous solutionat 1 mg/mL. The micelles of ssBC-100 had a hydrodynamicdiameter �35 nm, whereas those of ssBC-25 and ssBC-50containing thermoresponsive MEO2MA units had a smallerdiameter �29 nm with a small population of large aggre-gates (>1 mm). The occurrence of the large aggregation ispresumably attributed to the hydrophilic nature of ssBC-50and ssBC-25 copolymers.

An interesting observation of the above fluorescence meas-urements is the variation of fluorescence maximum wave-length (kmax) of NR encapsulated in hydrophobic cores ofssBCs with the different ratio of MEO2MA/HMssEt units. Asseen in Supporting Information Figure S5, the kmax was 612nm for ssBC-100, 615 nm for ssBC-50, and 619 nm for ssBC-25, and further 633 nm for ssBC-10; this result suggests thegreater kmax with an increasing amount of MEO2MA unit inthe cores. It is known that NR is a solvatochromic moleculewhose spectroscopic characteristics varies with polarity of alocal environment (typically, solvents).52 Here, the red shiftof the kmax value suggests the change of polarity inP(MEO2MA-co-HMssEt) cores to be more hydrophilic. Thiscan be attributed to the increasing amount of hydrophilicMEO2MA units in main chains.

FIGURE 3 DLS diagrams of 3 mg/mL of aqueous solution of ssBC-10 at 1 �C and 50 �C (a) and the variation of diameter through

reversible self-assembly and disassembly upon subsequent heating to 50 �C and cooling to 1 �C (b). [Color figure can be viewed

in the online issue, which is available at wileyonlinelibrary.com.]


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Studies of In Situ Disulfide-CrosslinkingThe resulting self-assembled amphiphilic micelles andtemperature-driven aggregates of ssBC copolymers containdifferent densities of pendant disulfide linkages. In the pres-ence of a catalytic amount of DTT, a typical reducing agent,

the occurrence of partial cleavage of the pendant disulfidelinkages results in intra- and interchain disulfide-thiolexchange reactions, leading to the formation of in situdisulfide-crosslinked nanogels. However, the use of excessDTT facilitates the complete cleavage of the disulfides to the

FIGURE 4 DLS diagrams of aqueous dispersion of ssBC-100 (a), ssBC-50 (b), and ssBC-25 (c) at 1 mg/mL at room temperature.

FIGURE 5 DLS diagrams of aqueous solutions of ssBC-100 micelles at 1 mg/mL in the presence of 0, 0.1, and 0.25 mol eq DTT to

disulfides before (a) and after dilution with 90% (b) and 95% (C) DMF.



corresponding thiols, leading to destabilization or disintegra-tion of aggregates. Because those micelles and aggregateshave different amounts of pendant disulfides, the use of theoptimized amount of DTT is critical to prepare colloidallystable, monodisperse nanogels.

First, amphiphilic micelles of ssBC-100, ssBC-50, and ssBC-25were examined. Aliquots of ssBC-100 micelles having onlyPHMssEt cores (DP of HMssEt5 49) were mixed with 0.1 and0.25 mol eq DTT to the disulfide linkages. After the mixtureswere stirred for 1 day, the efficacy of in situ disulfide cross-

linking was examined by mixing the resulting micellar disper-sions with excess DMF and followed by DLS measurements(Fig. 5). Note that DMF is a good solvent to both PEO andP(MEO2MA-co-HMssEt) chains. In the absence of DTT(DTT5 0 eq), the micelle size significantly decreased from�30 to <10 nm upon dilution with DMF. Such decrease sug-gests that the micelles are dissociated to polymeric chains inthe form of random coils in 90% DMF. In the presence of 0.1mol eq DTT, the micelle size increased to 60 nm in 90% DMF.However, upon further dilution to 95% DMF, it decreased to<12 nm. The decrease in size at 95% DMF is attributed to

FIGURE 6 DLS diagrams of aqueous solutions of ssBC-50 micelles treated with 0.5 mol eq DTT (a) and ssBC-25 micelles with 1

mol eq DTT after 3 days upon dilution with DMF.

FIGURE 7 DLS diagrams of aqueous solutions of ssBC-10 aggregates at 3 mg/mL at 30 �C (a, b) and 1 �C (c, d) in the presence of

difference amounts of DTT to be 0.5 (a, c) and 1.0 (b, d) mol eq to disulfides.


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incomplete disulfide-crosslinking. With a further increase inDTT to be 0.25 mol eq, the size increased to 46 nm in 90%DMF, and further to 72 nm in 95% DMF. The 0.25 mol eqDTT corresponds to 12 pendant disulfide linkages, suggestingthat �12 new disulfide crosslinks is sufficient for effectivecrosslinking through inter- and intra-disulfide-thiol exchangereactions to form nanogels of PEO-b-PHMssEt with DP5 49.

Both ssBC-50 and ssBC-25 micelles possess the DP of (MEO2-

MA1HMssEt) to be 49 and 44, similar to that (DP5 49) ofPHMssEt block for ssBC-100. However, they have lessHMssEt units in the P(MEO2MA1HMssEt) random copoly-mer block due to the lower feed ratio of HMssEt/MEO2MA.As shown in Table 1, the HMssEt units were 24 for ssBC-50and 11 for ssBC-25. While 0.25 mol eq DTT is sufficient forcrosslinking of ssBC-100, ssBC-50 micelles treated with 0.25mol eq DTT dissociated upon dilution with DMF. Accordingly,when the amount of DTT was increased to 0.5 mol eq todisulfides (roughly 12 disulfide crosslinks), a similar amountof in situ disulfide crosslinks as with 0.25 mol eq of DTT tossBC-100 can be generated. However, when 0.5 mol eq ofDTT was added to ssBC-50 micelles, after 1 day of crosslink-ing, they dissociated to individual polymer chains with diam-eter �12 nm in 90% DMF (Supporting Information FigureS6). After 3 days of crosslinking, they were stable in 95%DMF, as confirmed by DLS results with diameter �45 nm[Fig. 6(a)]. For ssBC-25 micelles, they were treated with 1.0mol eq DTT. Similar to ssBC-50 micelles, they dissociated in95% DMF after 1 day, but they were stable with diameter�47 nm after 3 days [Fig. 6(b)]. These results suggest thatat lower disulfide densities, a longer reduction reaction timeis required for effective in situ disulfide crosslinking.

Next, temperature-driven aggregation of ssBC-10 was exam-ined. To self-assembled aggregates prepared by heating to30�C in water, 0.5 and 1 mol eq of DTT to disulfides wereadded. The resulting mixtures were stirred at the sametemperature for 3 days. As seen in Figure 7a and b, the DLS

results indicate that with 0.5 and 1 mol eq of DTT at 30�C,the aggregates had diameters �23 nm, which was similarto that of physical aggregates without DTT. Next, the mix-tures were cooled to 5�C (below LCST) (Figure 7c and d).For the mixture with 0.5 eq DTT, the diameter decreased to�9 nm (Figure 7c), which is similar to that of ssBC-10 inaqueous solution at 1�C. The similar size to free polymerchains suggests lack of significant crosslinking. For the mix-ture with 1 mol eq DTT, however, the diameter remainedunchanged at �24 nm upon cooling to lower LCST (Figure7d). Figure 8 shows TEM images at different magnificationsof ssBC-10 crosslinked nanogels in the presence of 1 moleq DTT. Their analysis suggests the spherical morphologywith an average diameter5 28.56 11.5 nm. This result sug-gests the occurrence of crosslinking through the formationof new disulfide crosslinks, thus converting thetemperature-induced self-assembly aggregates to cross-linked nanogels. Further, the colloidal stability of the cross-linked nanogels was examined by mixing the resultingnanogel dispersions with DMF and followed by DLS meas-urements (Supporting Information Figure S7). In the pres-ence of 1 eq DTT, the nanogel size increased from 23 nm to43 nm upon dilution with 95% DMF, while the size distri-bution remained monomodal. The increase of size is attrib-uted to swelling of nanogels in a good solvent, while theoccurrence of mainly interchain disulfide-thiol polyexchangereactions in the PHMssEt cores prevents micelle dissocia-tion. A similar increase in size due to the occurrence ofcrosslinking reaction is reported for other core-crosslinkedmicelles.25,53,54

CONCLUSIONS

A series of new ssBC block copolymers of PEO-b-P(MEO2MA-co-HMssEt) having different densities of pendant disulfidelinkages were synthesized by ARGET ATRP. The amounts ofhydrophobic HMssEt units in the P(MEO2MA-co-HMssEt)random copolymer block controlled the aqueous

FIGURE 8 TEM images at different magnifications of ssBC-10 nanogels crosslinked in the presence of 1 eq DTT to disulfides. Scale

bar 5 100 nm.



characteristics of ssBCs to be amphiphilic or thermorespon-sive hydrophilic. Thus, they underwent amphiphilicity- ortemperature-driven self-assembly, yielding physically associ-ated aggregates in aqueous solution. For the occurrence ofin situ disulfide crosslinking through disulfide-thiol polyex-change reactions, the optimal concentration of DTT and lon-ger reaction time were important. Further, the formation ofsimilar densities of new disulfide linkages was required forthe conversion of physical aggregates with different den-sities of pendant disulfide linkages to the correspondingcrosslinked nanogels exhibiting enhanced colloidal stabilityupon dilution. These results of mutual relationship of disul-fide crosslinking density with morphological variation ofdisulfide-crosslinked nanogels could guide the furtherdevelopment of stimuli-responsive degradable nanogelsoffering enhanced colloidal stability and further controlleddrug release.

ACKNOWLEDGMENTS

This work is supported from NSERC Discovery Grant and Can-ada Research Chair (CRC) Award. NY thanks for NSERC Under-graduate Summer Research Award (USRA). JKO is entitled TierII CRC in Nanobioscience as well as a member of CentreQu�eb�ecois sur les Mat�eriaux Fonctionnels (CQMF) funded byFQRNT.

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Documents

Tuning amphiphilicity/temperature-induced self-assembly and in-situ disulfide crosslinking of reduction-responsive block copolymers