REVIEW www.rsc.org/softmatter | Soft Matter

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RAFT-synthesized diblock and triblock copolymers: thermally-inducedsupramolecular assembly in aqueous media†

Charles L. McCormick,*a Brent S. Sumerlin,*b Brad S. Lokitza and Jonathan E. Stempkaa

Received 19th December 2007, Accepted 21st April 2008

First published as an Advance Article on the web 4th July 2008

DOI: 10.1039/b719577j

This review highlights recent advances in the synthesis of functional, temperature-responsive,

water-soluble block copolymers, including particular focus on the results obtained by employing

reversible addition–fragmentation chain transfer (RAFT) polymerization. The applicability of the

RAFT process for the polymerization of functional monomers under a diverse range of experimental

conditions has facilitated the synthesis of water-soluble (co)polymers that were previously inaccessible.

Unprecedented control afforded by RAFT in homogeneous aqueous media allows well-defined

polymeric systems to be prepared without stringent purification techniques and under increasingly

‘‘green’’ conditions while maintaining the ability to tailor many of the macromolecular characteristics

(molecular weight, chain topology, copolymer composition, functionality, etc.) that affect

self-assembly in solution. Block copolymer formation and postpolymerization modification utilizing

crosslinking and copper-catalyzed azide–alkyne ‘‘click’’ chemistry are described, with attention

being paid to their ability to control copolymer structure for subsequent self-assembly in response to

changes in temperature.

Introduction

Although the utility of stimuli-responsive, synthetic (co)poly-

mers and biological macromolecules for speciality applications

has been widely recognized for over fifty years,2 only recently

have synthetic techniques become available that can yield

(co)polymers with the requisite architectures, molecular

Charles McCormick

CharlesMcCormick received his

PhD in chemistry from the

University of Florida. He is

currently a Bennett Distin-

guished Research Professor at

the University of Southern

Mississippi with appointments in

the Department of Polymer

Science and the Department of

Chemistry and Biochemistry.

His research focuses on stimuli-

responsive, water-soluble and

amphiphilic copolymers with

precisely designed architecture

prepared by controlled/‘‘living’’

free radical polymerization.

aDepartment of Polymer Science, University of Southern Mississippi,Hattiesburg, Mississippi, 39406, USA. E-mail: charles.mccormick@usm.edu; Fax: +1 601-266-5504; Tel: +1 601-266-4872bDepartment of Chemistry, Southern Methodist University, Dallas, Texas,75275-0314, USA. E-mail: bsumerlin@smu.edu; Fax: +1 214-768-4089;Tel: +1 214-768-8802

† Water-soluble polymers. Part 130.1

1760 | Soft Matter, 2008, 4, 1760–1773

weights, and narrow molecular weight distributions necessary

for specific technological applications. Among the most signi-

ficant are the controlled/living radical polymerization (CLRP)

techniques3 which include stable free radical polymerization

(SFRP),4 atom transfer radical polymerization (ATRP),5 and

reversible addition–fragmentation chain transfer (RAFT)

polymerization.6,7

Since initial reports by the CSIRO group in 1998,8,9 the RAFT

process has proven to be perhaps the most versatile of the CLRP

methods, since virtually all types of vinyl monomers can be

polymerized in bulk or with a variety of solvents under simple

reaction conditions. Recognizing the potential of the technique

for preparing homopolymers, block copolymers, and post-

reaction-modified polymers, several groups have successfully

Brent Sumerlin

Brent Sumerlin received his BS

from North Carolina State

University in 1998 and hisPhD in

Polymer Science and Engi-

neering from the University of

Southern Mississippi in 2003

under the direction of Prof.

Charles McCormick. He then

served as a visiting assistant

professor in the group of Prof.

Krzysztof Matyjaszewski at

Carnegie Mellon University. In

2005 he joined theDepartment of

Chemistry at Southern Metho-

dist University as an assistant professor where his research is

dedicated to responsive block copolyers and polymer–protein

conjugates.

This journal is ª The Royal Society of Chemistry 2008

Fig. 1 Selected water-soluble monomers polymerized via RAFT.

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prepared a range of water-soluble systems based on RAFT. Our

group reported many of the first examples of polymerization of

anionic, cationic, zwitterionic, and non-ionic monomers (Fig. 1)

under conditions, often directly in water, that required no

protecting groups.10 We have recently written a comprehensive

review detailing our work and that of a rapidly growing number

of other research groups concerning water-soluble (co)polymers

prepared via RAFT.11

In this review we focus on advances in the RAFT synthesis

of functional, hydrolytically-stable, water-soluble block

copolymers, including particular focus on the recent results

obtained in our laboratories. Specifically, we discuss synthetic

techniques for block copolymer formation as well as post-

polymerization modification utilizing crosslinking and ‘‘click’’

chemistry. We also discuss the analytical methodology often

utilized in ascertaining polymer structure and aqueous solution

behavior. We demonstrate that the facile control over block

copolymer structure allowed by RAFT is especially useful for

self-assembly in aqueous media in response to external stimuli.

We overview the self-assembly of selected block copolymers

into micelles and vesicles in response to changes in temperature

as well as the effects of crosslinking and/or postpolymerization

modification on the inherent properties of the resulting

materials. Based on the rapid pace of current RAFT

technology development, it is likely that stimuli-responsive

materials capable of being dissolved or dispersed in aqueous

media or coated onto surfaces as thin films will find unprece-

dented application in biomedical, pharmaceutical, optical, and

diagnostic areas.

This journal is ª The Royal Society of Chemistry 2008

Synthesis of block copolymers via RAFT

RAFT polymerization (Scheme 1) is an especially facile method

for preparing sequential blocks (e.g. AB, ABA, ABC) which may

serve directly to alter solution viscosity or be ‘‘triggered’’ by an

external stimulus such as pH, temperature, or ionic strength to

form supramolecular assemblies. Among the various methods of

CLRP, facile experimental setup and commercial availability of

most reagents required for ATRP has resulted in its being

employed in a majority of the literature reports concerning the

synthesis of amphiphilic block copolymers. However, despite

having been developed three years after the first reports of

ATRP, RAFT has increasingly been adopted for the preparation

of amphiphilic and responsive block copolymers that include

functional groups not easily accommodated by ATRP (e.g.,

–COOH, –SO3H, etc.). Additionally, the exceptional ability of

RAFT to control the polymerization of most acrylamido

monomers facilitates the preparation of a wide variety of well-

defined polymers that demonstrate temperature-responsive

solubility. Herein, our primary focus is the synthesis and

characterization of such temperature-responsive acrylamido

polymers prepared by RAFT polymerization.

The ability to form block copolymer structures via RAFT with

precise control of molecular weight, chain uniformity, and a,u

chain end functionality is a direct consequence of the use of

a chain transfer agent, or CTA. The role of the CTA (i) is to

suppress or limit the contribution of termination events that

occur during free radical polymerization. This suppression is

imposed through the establishment of an equilibrium between

Soft Matter, 2008, 4, 1760–1773 | 1761

Scheme 1 Mechanistic outline for RAFT homopolymerization (I) and block copolymerization (II).

Scheme 2 Idealized reversible aggregation in response to an external

stimulus.

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dormant (ii) and active chains (iv). The degenerative chain

transfer process proceeds through an intermediate radical species

(iii) which, once the main equilibrium has been established,

possesses polymeric segments. Fragmentation of the interme-

diate radical in either direction facilitates the uniform extension

of polymer chains. Although some aspects of the proposed

mechanism (for example the fate and lifetime of the intermediate

radicals)12 have not been resolved, proper selection of monomer,

CTA, initiator, and reaction conditions can yield impressive

results in terms of molecular weight control. Since the RAFT

process is ‘‘living’’, the reaction can be halted at predetermined

times and the polymer isolated. The resultant CTA-functional-

ized polymer can then serve as a macroCTA (v) for block

copolymer formation utilizing a second monomer in a manner

analogous to that of homopolymerization. Further extension of

the diblock macroCTA (vi) with a third monomer, yielding

a controlled triblock, can also be accomplished.

A critical consideration in preparation of responsive polymers

discussed in this review is control of segmental molecular weight

(and molecular weight distribution) during homopolymerization

(Scheme 1, I), diblock copolymerization (Scheme 1, II), or higher

extension, for example to form triblocks. Under appropriate

RAFT conditions, theoretical molecular weight, Mn,Th, can be

estimated with eqn (1). Thus, segmental length can be targeted

for each block by simply controlling the [monomer]0:[CTA]0ratio and the conversion since the respective molecular weights of

the monomer, MWmon, and CTA, MWCTA, are known.

Mn;Th ¼�½monomer�0�MWmon

½CTA�0� conversion

�þ MWCTA (1)

Stimuli-responsive block copolymers via RAFT

Stimuli-responsive block copolymers typically contain both

permanently hydrophilic blocks and ‘‘smart’’ blocks which are

tunably hydrophilic/hydrophobic.13,14 The stimuli-responsive

block copolymers undergo conformational changes in response

to changes in external stimuli such as pH, electrolyte concen-

tration, and/or temperature. The changes can cause the ‘‘smart’’

1762 | Soft Matter, 2008, 4, 1760–1773

blocks to become hydrophobic and induce the self-assembly of

the amphiphilic block copolymer into supramolecular structures,

such as micelles and vesicles (Scheme 2).10,11,15–19

Temperature-responsive block copolymers via RAFT

The most common temperature-responsive polymers are prepa-

red from N-alkyl acrylamide monomers. Of these, poly(N-iso-

propylacrylamide) (PNIPAM) from 16 (Fig. 1) has received the

most attention due to its lower critical solution temperature

(LCST) of �32 �C in water.20 With the temperature of the

physiological fluids within the human body being 37 �C, NIPAM

copolymers, including those with crosslinked modification, have

been targeted for drug delivery applications.21–23

To date there have been numerous reports detailing the

successful RAFT polymerization of NIPAM in organic

solvents.14 For example, Ganachaud et al.24 reported the AIBN-

initiated solution polymerization of NIPAM employing both

benzyl dithiobenzoate (in benzene) and cumyl dithiobenzoate (in

1,4-dioxane) at 60 �C. Subsequently, Schilli et al.25 disclosed the

benzyl and cumyl dithiocarbamate-mediated polymerization of

NIPAM, also in 1,4-dioxane at 60 �C. These experimental

conditions led to polymers with polydispersity indices (PDIs)

around 1.3. Winnik and coworkers have demonstrated the

polymerization of NIPAM using a variety of trithiocarbonates

and have recently investigated areas such as end group associa-

tion,26 mesoglobule formation,27 and chain end modification.28

More recently, Ray and coworkers29 demonstrated the ability to

control the tacticity in RAFT polymerizations of NIPAM via the

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addition of a suitable Lewis acid such as Sc(OTf)3 or Y(OTf)3.

RAFT-prepared PNIPAM has also been employed as a thermo-

responsive stabilizing layer for gold nanoparticles/clusters30–32

following a synthetic procedure we reported earlier for macro-

CTA grafting onto noble metals.33,34

RAFT has also been utilized to synthesize NIPAM-based

block copolymers. Yusa and Morishima35 studied the thermo-

responsive aggregation of the block copolymer poly[2-(acryl-

amido)-2-methylpropanesulfonate (NaAMPS) (3)-b-NIPAM,

Virtanen et al.36 studied the sequestration of fluorescent probes by

PEO-b-NIPAM micelles, Liu and Perrier37 prepared block

copolymers of DMA (15) and NIPAM in 1,4-dioxane, and

Arotcarena and coworkers38,39 prepared doubly thermorespon-

sive block copolymers of 3-ammoniopropane sulfonate (SPP) and

NIPAM. More recently, Voit et al.40 demonstrated the extension

of NIPAM macroCTAs with a number of glycomonomers,

resulting in sugar-containing responsive block copolymers. The

comonomer content, glycomonomer spacer length, and chain

Fig. 2 Selected RAFT chain transfer agents (CTAs) and azo-initiators.

Fig. 3 a) Pseudo-first-order kinetic plot and b) Mn and c) Mw/Mn versus conv

CTA 1 or CTA 2 at 25 �C using I3 as a radical source. Monomer conversion a

and size exclusion chromatography (SEC), respectively. Adapted with permis

This journal is ª The Royal Society of Chemistry 2008

architecture were shown to have a dramatic impact on the cloud

points of the copolymers. Oupicky and coworkers41 utilized

RAFT to synthesize heterobifunctional block copolymers of

PEG-b-NIPAM with an internal lysine residue at the focal point

and a terminal thiol group which was used to conjugate biotin.

The copolymers demonstrated temperature-induced association

and formed complexes with avadin.

The following sections focus on recent advances in the RAFT

polymerization of temperature-responsive polymers reported by

our respective research groups (McCormick at USM and

Sumerlin at SMU) and several others within the field. Specifically,

key elements of the aqueous RAFT polymerization of NIPAM

and the synthesis of thermally responsive block copolymers

directly in water,42,43 the facile preparation of temperature-

responsive shell cross-linked (SCL) micelles,44,45 and the prepa-

ration of thermally responsive hyperbranches through the

combination of RAFT and click chemistry46 will be presented.

Thermally responsive block copolymers of containing

N-isopropylacrylamide (16)

While the RAFT polymerization of a diverse variety of mono-

mers directly in water had previously been reported, the LCST of

PNIPAM had generally been considered to preclude the possi-

bility of living polymerization in homogeneous aqueous media

because of the potential for precipitation at the high temperatures

generally employed for RAFT. Thus, an important step toward

the synthesis of thermally responsive, water-soluble block

copolymers was accomplished when Convertine et al. conducted

the room temperature RAFT polymerization of NIPAM directly

in water.47,48 Employing an azo-initiator with an appropriately

short half-life and the CTAs shown in Fig. 2 allowed controlled/

living polymerization of NIPAM, as evidenced by pseudo-first-

order kinetics, linear increase in molecular weight with conver-

sion, and narrow molecular weight distributions (Fig. 3).

The success of the controlled room temperature polymeriza-

tion of NIPAM has allowed for the aqueous synthesis of a

series of thermally responsive AB diblock and ABA triblock

ersion for the aqueous homopolymerization of NIPAM (16) mediated by

nd molecular weight distribution were determined via NMR spectroscopy

sion from ref. 42. Copyright 2006 American Chemical Society.

Soft Matter, 2008, 4, 1760–1773 | 1763

Fig. 5 Temperature-induced reversible association of P(DMA100

NIPAM460) block copolymers as measured by DLS upon cycling between

25 and 45 �C at 30 min intervals. Adapted with permission from ref. 42.

Copyright 2006 American Chemical Society.

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copolymers.42 These polymers contained hydrophilic DMA A

blocks of fixed molecular weight and temperature-responsive

NIPAM B blocks of varied chain length. These thermally

responsive, water-soluble block copolymers were capable of

reversibly forming micelles in response to changes in solution

temperature, and the micelle size and transition temperature

were dependent on both the NIPAM block length and the

polymer architecture (diblock vs. triblock). Mono- and difunc-

tional macroCTAs of DMA prepared from CTA 1 and CTA 2

(Fig. 2), respectively, were used for the chain extension of

NIPAM in water at 25 �C to yield a range of AB diblock and

ABA triblock copolymers (Scheme 3). The self-assembly of the

thermally responsive, water-soluble block copolymers was

followed by dynamic light scattering (DLS). Above the LCST,

PNIPAM chains become dehydrated due to an entropy gain

resulting from the release of water molecules upon association of

the isopropyl groups.20,49 A reversible transition from mole-

cularly dissolved unimers to aggregated micelles occurred above

the critical micelle temperature (CMT).

Insight into association behavior was obtained by examining

DLS data of the responsive block copolymers as a function of

temperature and by static light scattering by estimating the

number of unimers constituting the respective micelles.42 As

shown in Fig. 4, increased NIPAM segment length led to lower

CMTs and aggregates with larger hydrodynamic diameters.

Scheme 3 Synthetic route for preparation of diblock copolymers of

DMA and NIPAM via aqueous room temperature RAFT.

Fig. 4 Hydrodynamic diameter (Dh) as a function of temperature for

a series of diblock copolymers, as measured by dynamic light scattering.

Adapted with permission from ref. 42. Copyright 2006 American

Chemical Society.

1764 | Soft Matter, 2008, 4, 1760–1773

Interesting time-dependent reorganization (compaction) of

structure was evident for intermediate block lengths. With

sufficient time for reorganization, cycling between the unimeric

(25 �C) and assembled (45 �C) states was realized for the

P(DMA100-b-NIPAM460) block copolymers (Fig. 5).

In contrast to ABA triblock copolymers, BAB triblock

copolymers can form flower micelles at low concentrations and

physical gels at moderate to high concentrations (Fig. 6). Armes

et al. have reported the synthesis of several temperature- and

pH-responsive triblock copolymers utilizing ATRP.50–54 Several

of the copolymer gels have potential applications as controlled

release substrates53 and cell growth scaffolds.52 Recently, we

reported the aqueous RAFT polymerization of BAB triblock

copolymers, where the B blocks consist of PNIPAM and the A

block consists of PDMA.55 At moderate to high concentrations,

the triblock copolymers P(NIPAM455-DMA210-b-NIPAM455)

and P(NIPAM455-DMA277-b-NIPAM455) could form physical

Fig. 6 The formation of flower micelles and physical gels from BAB

triblock copolymers.

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gels under physiological conditions (140 mM NaCl/20 mM

phosphate buffer, pH 7.4, 37 �C). In addition, the mechanical

properties were similar to collagen, a commonly used cell growth

platform.55

Muller and Barner-Kowollik et al. reported another example

of responsive systems based on PNIPAM being prepared directly

in homogeneous aqueous media. Block copolymers of NIPAM

and acrylic acid (AA) were prepared by aqueous RAFT initiated

with g-irradiation.56 The temperature-responsive nature of

the PNIPAM block, coupled with the pH-responsive charac-

teristics of the poly(acrylic acid) (PAA), give rise to block

copolymers with potential doubly responsive or ‘‘schizophrenic’’

behavior.57,58 RAFT has also been utilized for the synthesis of

block copolymers with a PNIPAM segment and a second

segment demonstrating the opposite temperature response.59

Because the second block was composed of zwitterionic repeat

units, it demonstrated upper critical solution temperature

(UCST) aqueous solution behavior that facilitated the prepara-

tion of micelles with PNIPAM coronas at low temperatures.

ABC triblock copolymers incorporating the active monomer

N-acryloxysuccinimide (18) and the formation of shell

cross-linked (SCL) micelles

When solutions of polymer micelles are diluted below the critical

micelle concentration (CMC), dissociation to unimers occurs.

Therefore, under conditions of high dilution (e.g., in vivo),

polymeric micelle stability is compromised, and the potential for

controlled-delivery applications is reduced. To circumvent this

limitation, several groups have explored stabilization of the

micelle corona through chemical or physical cross-linking. These

stabilized micelles, commonly referred to as shell cross-linked

(SCL) micelles, were first reported by Wooley and coworkers60 in

1996 and are the subject of a recent review by Armes et al.61 SCL

micelles have potential applications in drug delivery, emulsifi-

cation, sequestration of metabolites, and entrapment of

environmental pollutants.62–68 Recently, we44 synthesized ABC

triblock copolymers of poly(ethylene oxide) (PEO)-b-P((DMA-

stat-N-acryloxysuccinimide (NAS))-b-NIPAM) (Scheme 4) and

Scheme 4 Pathway for the synthesis of the PEO-b-(DMA-s-NAS)-b-

NIPAM triblock copolymers.

This journal is ª The Royal Society of Chemistry 2008

demonstrated the facile formation of SCL micelles. One hydro-

philic segment of the ABC triblock copolymer was comprised of

PEO, selected on the basis of demonstrated biocompatibility and

dual solubility in both aqueous and organic media. Incorpora-

tion of PEO segments into block copolymers prepared by RAFT

is generally accomplished by first preparing a macroCTA from

u-hydroxy PEO.69,70 Polymerization in the presence of such

a RAFT agent allows in situ block copolymer formation. A

procedure similar to that reported by Perrier and coworkers was

employed to prepare the PEO macroCTA necessary for synthesis

of PEO-b-P((DMA-stat-NAS)-b-NIPAM) (Scheme 4).71 NAS

units in the resulting block copolymer demonstrated minimal

susceptibility to hydrolysis and served as internal crosslinking

sites for reaction with difunctional primary amines.72 The

thermoresponsive block of the copolymer was prepared by

RAFT polymerization of NIPAM.

The aqueous self-assembly behavior of these NIPAM-based

triblock copolymers was investigated73 utilizing DLS (Fig. 7).

The CMT for the block copolymers decreased with increasing

NIPAM block length, consistent with previous reports.43,48,74

In contrast to the previously discussed P(DMA-b-NIPAM)

copolymers, hydrodynamic dimensions were also dependent on

the NIPAM/NAS ratio within the middle block, as typically

observed for statistical copolymers with hydrophilic and

hydrophobic segments.75

Incorporation of NAS provided a facile and efficient strategy

for formation of SCL micelles. This was accomplished by addi-

tion of a diamine to the aqueous micelle solution (Scheme 5). The

cross-linking of the hydrophilic shell with ethylenediamine was

rapid, resulting in over 95% completion within 2 h. Notably,

Wooley and coworkers76 adopted this cross-linking procedure to

prepare SCL micelles based on the amphiphilic block copolymer

poly((methyl acrylate)-b-(NAS-co-(N-acryloylmorpholine))). In

our studies, aggregate structure of the SCL micelles was

conserved after reduction of the solution temperature; instead of

dissolution to unimers, the NIPAM cores swelled after being

rendered hydrophilic below the CMT. Atomic force microscopy

(AFM) images of the SCL micelles prepared from an example of

this triblock showed the spherical shape and relative uniformity

of the micelles (Fig. 8).

Fig. 7 Hydrodynamic diameter vs. temperature for the PEO-b-

P((DMA-stat-NAS)-b-NIPAM) triblock copolymers in aqueous solu-

tion. Adapted with permission from ref. 73. Copyright 2006 American

Chemical Society.

Soft Matter, 2008, 4, 1760–1773 | 1765

Scheme 5 Self-assembly into micelles and shell cross-linked micelles of PEO-b-P((DMA-stat-NAS)-b-NIPAM) triblock copolymers. Adapted with

permission from ref. 73. Copyright 2006 American Chemical Society.

Fig. 8 Tapping-mode AFM images of PEO-b-P((DMA-s-NAS)-b-

NIPAM) micelles after cross-linking with ethylenediamine. (A) Height

image; (B) Phase image. Samples were prepared by drop deposition (5 mL,

0.01% concentration) onto freshly cleaved mica and allowed to dry in air.

Adapted with permission from ref. 73. Copyright 2006 American

Chemical Society.

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Reversible SCL micelles and model compound release

A major concern for drug delivery with SCL micelles is the

possibility of in vivo accumulation of non-degradable materials in

the kidneys and other organs. One approach to overcome this

problem is to provide chemical or physical cross-linking entities

functionally labile in the physiological environment. To

demonstrate the feasibility of such a process, micelle-forming

triblocks of PEO45-b-P((DMA98-stat-NAS30)-b-NIPAM87) were

prepared by a procedure similar to that described in the previous

Scheme 6 Formation of reversible shell cross-linked micelles from PEO-b

cystamine. Adapted with permission from ref. 77. Copyright 2006 American

1766 | Soft Matter, 2008, 4, 1760–1773

section.77 After heating a solution of the block copolymer to

45 �C (above the CMT, 37 �C), the resulting micelles were cross-

linked with cystamine, a disulfide-containing diamine, in an

equimolar ratio with the NAS units in the statistical block

(Scheme 6).

Disulfide bonds can be readily cleaved using a thiol-exchange

reaction with dithiol compounds such as dithiothreitol (DTT)78,79

or tris(2-carboxyethyl)phosphine hydrochloride (TCEP).80 When

DTT was used to cleave the disulfide bonds at 45 �C, complete

reaction was achieved within 10 h. TCEP, a more efficient

reducing agent, led to complete cleavage within 30 min. In both

cases unimer formation was confirmed by DLS and by SEC.

After removal of excess reducing agent from the solution of

cleaved micelles, addition of cystamine caused reconstitution of

the SCL structure via the resulting thiol/disulfide exchange

reaction. Fig. 9 shows the respective DLS size distributions for

PEO45-b-(DMA98-s-NAS30)-b-NIPAM87 unimers at 25 �C (peak

1), micelles at 45 �C (peak 2), swollen SCL micelles at 25 �C (peak

3), unimers at 25 �C after cleavage with DTT (peak 4), micelles at

45 �C after cleavage with DTT (peak 5), and SCL micelles after

a second cross-linking reaction with cystamine at 25 �C (peak 6).

The drug delivery potential of the reversible SCL micelles was

assessed by release of a model drug, dipydridamole (DIP). DIP

was loaded into the hydrophobic micelle core at 45 �C. Lowering

the solution temperature to 25 �C caused the micelles to

dissociate into unimers and resulted in burst release of the drug,

as monitored by UV absorption of DIP at 415 nm (Fig. 10).

Cross-linking the micelle core with cystamine led to significant

retardation of release, both at 25 and 45 �C. These results suggest

that drug-release rate can be tuned by the degree of cross-linking.

-P((DMA-stat-NAS)-b-NIPAM) triblock copolymers by reaction with

Chemical Society.

This journal is ª The Royal Society of Chemistry 2008

Fig. 9 Size distribution of 0.5 % aqueous solution of PEO45-b-

P((DMA98-s-NAS30)-b-NIPAM87) at (1) 25 �C; (2) 45 �C; (3) SCL

micelles at 25 �C; (4) SCL micelles at 25 �C after cleavage with DTT; (5)

SCL micelles at 45 �C after cleavage with DTT; (6) SCL micelles at 25 �C

after cross-linking the cleaved micelles with cystamine. Adapted with

permission from ref. 77. Copyright 2006 American Chemical Society.

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While leading to irreversible cleavage, H2O2 can also be used to

degrade the micelle cross-links.81 Because H2O2 is produced in

mammalian immune systems,82 SCL micelle cleavage in situ

could potentially facilitate return to unimers and subsequent

elimination from the body.

SCL micelles from block copolymers containing an unprotected

amino acid based monomer

McCormick83–86 and others87–90 have reported the polymerization

of N-acryloyl derivatives of amino acids to obtain a variety of

water-soluble polymers. These monomers can be synthesized in

a facile manner from readily available amino acids, and their

amphiphilic nature allows for polymerization, purification, and

characterization directly in aqueous solution. In addition, the

chiral structures produced from polymerization of the respective

D and L enantiomeric monomers have potential optical and

pharmaceutical applications.

Fig. 10 Cumulative DIP release to PBS buffer from (a) shell cross-linked (SC

the presence of DTT (B) and without DTT (-). Adapted with permission f

This journal is ª The Royal Society of Chemistry 2008

The RAFT polymerization of AAL (6) based on L-alanine was

accomplished directly in water using initiator I3 and CTA 1 and

CTA 2 (Fig. 2) and proceeded in a controlled manner as

evidenced by low polydispersities and linear increases in mole-

cular weight with conversion.43 Having established conditions for

controlled/living homopolymerization, thermally responsive

triblock and pentablock copolymers with NIPAM and DMA

were prepared (Scheme 7). The initial hydrophilic, neutral block

was synthesized from DMA with a monofunctional (Pathway A)

or difunctional (Pathway B) CTA. Sequential block copolymer-

ization with AAL followed by NIPAM yielded well-defined block

copolymers that were subsequently characterized in solution.

For both the tri- and pentablock copolymers, raising the

solution temperature above the CMT led to a transition from

molecularly dissolved unimers to micelles. Fig. 11 shows the

temperature-induced changes in the hydrodynamic volume for

block copolymers with varying composition. As the PNIPAM

block length increased, CMT decreased and average aggregate

size increased, as described earlier.48

Block copolymers with AAL units also allowed the formation

of SCL micelles. The presence of the anionic carboxylate groups

within the AAL blocks of the thermally-assembled micelles

allowed ionic crosslinking91 (Scheme 8) through the addition of

an equimolar (by repeat unit) amount of poly((ar-vinylbenzyl)

trimethylammonium chloride) PVBTAC (9). Electrostatic inter-

action between the coronal AAL polyanion blocks and the

polycation unimers led to interpolyelectrolyte cross-linked

micelles that remained intact at reduced temperatures. The slight

decrease in micelle size observed when the solution was cooled to

room temperature was attributed to reduced electrostatic repul-

sion of the carboxyl groups, facilitating more efficient packing of

the AAL segments.

The self-assembled morphology of the block copolymers has

been confirmed by transmission electron microscopy (TEM)

which shows interpolyelectrolyte cross-linked micelles with

diameters between 30 and 40 nm, in reasonable agreement with

that of 34 nm determined by DLS.

The stability/reversibility of the ionically SCL micelles was

investigated by introducing simple electrolytes (Fig. 12). Micelles

L) and un-cross-linked micelles at 25 �C and (b) SCL micelles at 37 �C in

rom ref. 77. Copyright 2006 American Chemical Society.

Soft Matter, 2008, 4, 1760–1773 | 1767

Fig. 11 Apparent hydrodynamic diameters (Dh) for the block copoly-

mers measured by dynamic light scattering (1.0 g L�1) as a function of

temperature. Adapted with permission from ref. 43. Copyright 2006

American Chemical Society.

Scheme 8 Temperature-responsive micellization of block copolymers

and reversible interpolyelectrolyte-complexed micelle formation.43

Fig. 12 Apparent hydrodynamic diameters (Dh) as a function of sodium

chloride concentration ([NaCl]) for ionically cross-linked DMA100-b-

AAL65-b-NIPAM165 triblock copolymer micelles.43

Scheme 7 Synthetic routes for preparation of ABC (Pathway A) and ABCBA (Pathway B) block copolymers via aqueous RAFT.

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remained intact in water at NaCl concentrations as high as

0.3 M; the dissociation into unimers was observed at a NaCl

concentration of 0.4 M. Thus, dissolution of the aggregates

demonstrated the reversibility of the interpolyelectrolyte

1768 | Soft Matter, 2008, 4, 1760–1773

crosslinks. Interestingly, above 0.8 M NaCl, aggregates were

reformed, as the NIPAM block was ‘‘salted out.’’ The facility of

formation and the reversibility of these interpolyelectrolyte-

complexed micelles suggest the possible utility of such structures

in pharmaceutical applications.

Vesicles from thermally responsive block copolymers

Vesicles composed of lipid molecules play important roles in

several biological functions, including the storage and trans-

portation of small molecules.92,93 Vesicles prepared from block

copolymers or ‘‘polymersomes’’ have been extensively studied for

biological applications due to possible increased integrity arising

from intermolecular chain entanglements.94–97 Methods of vesicle

formation from typical amphiphilic block copolymers involve

the use of organic cosolvents such as THF,96 DMF,94 or

dioxane.98 Such polymersome solutions generally require exten-

sive purification processes which can be time-consuming and

This journal is ª The Royal Society of Chemistry 2008

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problematic. A more versatile route is the stimuli-responsive

self-assembly of asymmetric block copolymers in water.99–102

Polymersomes were conveniently prepared by the self-

assembly of poly(N-(3-aminopropyl) methacrylamide hydro-

chloride (12))-b-NIPAM) block copolymers directly in water

(Scheme 9).45 These diblock copolymers readily dissolved in

aqueous solution at room temperature. Increasing the solution

temperature above the LCST of the NIPAM block led to

uniform aggregates with diameters of approximately 280 nm.

The large, uniform sizes and TEM images are indicative of

polymersomes (Fig. 13). Increasing the hydrophilic AMPA block

length while keeping the NIPAM block length constant resulted

in an increase in the phase transition temperature.45 A similar

effect was observed by Xia et al. for NIPAM homopolymers with

differing end group hydrophilicites.103

A sufficiently slow heating rate (0.1 �C min�1) during the

morphological transition from unimers to vesicles was needed in

order to obtain aggregates with uniform size. Once above the

transition temperature, polymersome size remained constant,

suggesting self-assembly was kinetically controlled. Solution

concentration also affects size, as relatively low block copolymer

concentrations (<0.5 mg ml�1) led to more uniform size distri-

butions. Since AMPA is pH-responsive, the stability of the

polymersomes between pH 0 and 11 was investigated. Over this

Scheme 9 Formation of vesicles from PAPMA-PNIPAM diblock

copolymers and their subsequent ionic cross-linking. PAPMA: poly(N-

(3-aminopropyl) methacrylamide hydrochloride). Copyright Wiley-VCH

Verlag GmbH & Co. KGaA. Reproduced with permission from ref. 45.

Fig. 13 Transmission electron microscopy images of (A) vesicles

prepared from PAMPA88-PNIPAM50 via rapid increase of solution

temperature from 25 �C to 45 �C (magnification 25k). (B) Single vesicle

(magnification 50k). Copyright Wiley-VCH Verlag GmbH & Co. KGaA.

Reproduced with permission from ref. 45.

This journal is ª The Royal Society of Chemistry 2008

range, the structures remained intact, while varying in size with

solution pH. The changes in size (310 nm at pH 3.0 and 220 at pH

10.8) were consistent with the expected degree of protonation of

the AMPA units.

Interpolyelectrolyte complexation can also be used to ‘‘fix’’ or

cross-link the shells of the polymersomes. Addition of an anionic

polyelectrolyte, poly(sodium 2-acrylamido-2-methylpropane-

sulfonate) (AMPS, 3) to a solution of PAPMA-PNIPAM

diblock copolymer resulted in stabilized ‘‘crosslinked’’ structures.

Upon introduction of the oppositely charged AMPS homo-

polymer, vesicle size decreased from 270 to 140 nm due to charge

neutralization upon complexation. After cross-linking, the

solution temperature was reduced to 25 �C without loss in vesicle

integrity. The resulting ionically cross-linked vesicles were

stable over a wide pH range and moderate electrolyte concen-

tration. Raising the electrolyte concentration above 0.8 M NaCl

caused vesicle dissociation, thereby demonstrating cross-link

reversibility.

Another method for the ‘‘fixing’’ of the shell of polymersomes

has recently been reported.104 Gold nanoparticle-decorated

polymersomes can be synthesized by mixing a solution of

poly[2-dimethylamino)ethyl methacrylate (13)-b-(N-isopropyl-

acrylamide) with a solution of NaAuCl4 above the LCST

(Scheme 10). The formation of gold nanoparticles in the

PDMAEMA domains functions as a cross-linking agent due to

the anchoring of multiple chains to the surface of the nano-

particles. In Fig. 14, the transition from unimers at 25 �C (a) with

hydrodynamic diameter (Dh) below 8 nm to polymersomes (b)

with average Dh of 140 nm. After formation of the gold nano-

particles, the polymersome size and size distribution at 50 �C

increased slightly (b to c), which is attributed to increased

protonation of the PDMAEMA segments during equilibration

and gold complex reduction. When the gold nanoparticle-

decorated polymersomes are cooled to 25 �C, the polymersomes

remain intact and the size again increases due to the increased

hydrophilicity of the PNIPAM block.

RAFT and click chemistry

Clearly, the efficiency provided by CLRP allows control of

molecular weight, molecular weight distribution, and chain end

functionality and has served to significantly advance the field of

controlled architecture polymers. The versatility of RAFT

polymerization and its utility in aqueous media facilitates pre-

paration of stimuli-responsive block copolymers that were only

recently considered inaccessible. In addition to the functional

group tolerance and efficiency inherent with this technique,

postpolymerization modification remains a valuable tool by

Scheme 10 Formation of thermally responsive vesicles decorated with

gold nanoparticles. Adapted with permission from ref. 104. Copyright

2007 American Chemical Society.

Soft Matter, 2008, 4, 1760–1773 | 1769

Fig. 14 Dynamic light scattering size distribution of a 0.01 wt%

PDMAEMA73-b-PNIPAM99 diblock copolymer solution: a) 25 �C; b) 50�C; c) 50 �C after in situ reduction of NaAuCl4; d) after in situ reduction

of NaAuCl4 upon lowering temperature to 25 �C. Adapted with

permission from ref. 104. Copyright 2007 American Chemical Society.

Fig. 15 Structures of novel azido CTAs employed to prepare functional

telechelic polymers by RAFT.

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which additional functionality may be incorporated. In parti-

cular, transformation of polymer end groups is a useful method

for preparation of surface-immobilized polymers,33,34 fluores-

cently-labeled chains,105–107 and bioconjugates.108,109 The inherent

low concentration of end groups and the possibility of side

reactions with other functional groups contained in the polymer

require reactions with high efficiency and fidelity for successful

and specific polymer modification.

Recently developed synthetic techniques have demonstrated

great promise for precise polymer functionalization. CuI-cataly-

zed azide–alkyne coupling (CuAAC) (Scheme 11) results in

highly specific and efficient preparation of 1,4-disubstituted

1,2,3-triazole products.110,111 This particular coupling process can

be conducted under moderate reaction conditions, in aqueous or

organic media, and with few or no side reactions. The practicality

and versatility of CuAAC has led to its inclusion in the class of

efficient and specific organic reactions, commonly termed ‘‘click’’

chemistry, as coined by Sharpless et al.112

Several groups have reported the synthesis of highly functional

(co)polymers by CLRP and subsequent azide–alkyne coupling

reactions, although these reports predominately concern the

modification of (co)polymers prepared by ATRP or SFRP.113–121

For instance, we reported the preparation of u-(meth)acryloyl

macromonomers via ATRP and CuAAC.122 While this approach

proved an efficient means to prepare highly branched polymers

from any monomer polymerizable by ATRP, we aimed to

expand the method to other monomer classes polymerizable by

RAFT.

Previously, Hawker and Wooley et al. reported the RAFT

block copolymerization of a protected acetylene-containing

monomer.123 After deprotection, the resulting block polymers

were employed to prepare shell-crosslinked micelles with cores

Scheme 11 General scheme for CuI-catalyzed azide–alkyne coupling.

1770 | Soft Matter, 2008, 4, 1760–1773

susceptible to functionalization with low molecular weight

azides. The same authors reported alkynyl-functionalized RAFT

agents for preparation of surface-decorated micelles that were

reacted with azido compounds.124

CuAAC has also been employed for end group functionali-

zation of RAFT-derived polymers.46,125,126 We prepared two

azido-functionalized CTAs, namely 2-dodecylsulfanylthio-

carbonylsulfanyl-2-methylpropionic acid 3-azidopropyl ester

(CTA 3) and 4-cyano-4-methyl-4-(thiobenzoylsulfanyl)butyric

acid 3-azidopropyl ester (CTA 4) (Fig. 15). These novel

compounds were used to prepare PDMA homo- and block

copolymers that were subsequently functionalized with a variety

of low molecular weight alkynes.46 Preparing end functional

polymers or functional CTAs by modification via alternative

reaction pathways might have less applicability due to limited

efficiency and orthogonality. However, the fidelity associated

with click chemistry allows facile preparation of a range of

functional macromolecules.

Postpolymerization modification of temperature-responsive

(co)polymers by CuAAC has allowed us to prepare a range of

functional systems with biological relevance. For instance,

azido-terminated P(DMA-b-NIPAM) was efficiently coupled

with propargyl folate to yield a diblock copolymer capable of

temperature-responsive self-assembly in aqueous media

(Scheme 12).127 Because the folate residue was incorporated on

the end group of the hydrophilic PDMA block, the resulting

micelles were candidates for tumor-specific drug delivery.

Scheme 12 Folate-terminated P(DMA-b-NIPAM) block copolymers

and polymeric micelles.127

This journal is ª The Royal Society of Chemistry 2008

Scheme 13 Polymer–protein bioconjugates prepared by CuAAC of alkyne-functionalized bovine serum albumin (BSA) and azido-terminated PNI-

PAM.128

Fig. 16 Hydrodynamic diameter (Dh) versus temperature for a 5% w/v

aqueous solution of highly branched PNIPAM (11 000 g mol�1, 4%

branching). Adapted with permission from ref. 46. Copyright 2007

CSIRO.

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Azido-terminated polymers prepared by RAFT can also be

readily conjugated to biomacromolecules, as we recently

demonstrated. PNIPAM-N3 was efficiently coupled with a model

alkyne-functionalized protein by CuAAC.128 Bovine serum

albumin was labeled with an alkyne by reaction of its lone free

cysteine with propargyl maleimide. The resulting activated

protein was coupled with azido-terminated PNIPAM to yield

well-defined polymer–protein bioconjugates capable of tempe-

rature-responsive self-assembly (Scheme 13).

CuAAC has also proven to be a feasible method for

functionalization of low molecular weight CTAs prior to poly-

merization. Whereas other methods of CTA modification are

limited because of potential side reactions with the thiocarbonyl

or other susceptible moieties, the orthogonal nature of click

chemistry facilitates specific CTA functionalization. Recently,

a CTA prepared by this method was utilized to synthesize

hyperbranched PNIPAM.46 CTA 3 was reacted with propargyl

acrylate to yield an acryloyl trithiocarbonate (Scheme 14,

CTA 4) that contained both monomer and CTA functionality.

Homopolymerization of AB* monomers containing both

a polymerizable double bond (A) and an initiating moiety (B*)

can lead to hyperbranched polymers via a process often termed

self-condensing vinyl polymerization (SCVP).129 Accordingly, the

acryloyl trithiocarbonate was copolymerized with NIPAM to

yield thermoresponsive hyperbranches. By virtue of the RAFT

process, the individual branches were well-defined with length

and number depending on the ratio of [NIPAM]:[CTA 4], and

each chain end contained the dodecyltrithiocarbonate moiety

derived from the original CTA.

The aqueous thermoresponsive nature of these copolymers

was investigated since highly branched polymers often demon-

strate significantly different solubility behaviors from their linear

counterparts. In addition to reduced chain entanglement, end

Scheme 14 Synthesis of an acryloyl trithiocarbonate chain transfer

agent (CTA 4) and subsequent branched PNIPAM preparation via

RAFT. Adapted with permission from ref. 46. Copyright 2007 CSIRO.

This journal is ª The Royal Society of Chemistry 2008

group effects can be particularly important since branched

polymers contain multiple termini.130 We observed transition

temperatures significantly lower than the typical values. For

example, PNIPAM (Mn ¼ 11 000 g mol�1, 4% branching)

prepared with CTA 4 showed a dramatic increase in hydro-

dynamic diameter, indicative of intermolecular aggregation at

just 25 �C (Fig. 16). As the degree of branching increased, the

LCST of the hyperbranched polymer decreased. For instance,

PNIPAM with 10% branching was soluble in water only below

2 �C, an observation attributed to the enhanced hydrophobic

contributions of the dodecyl chain termini. Indeed, end group

cleavage by radical induced reduction yielded PNIPAM hyper-

branches with hydrogen termini and the expected LCST of 32 �C.

4. Conclusion/future work

The applicability of the RAFT process for the polymerization of

functional monomers under a diverse range of experimental

conditions has facilitated the synthesis of responsive, hydrolyti-

cally-stable, water-soluble (co)polymers that were previously

inaccessible. Unprecedented control afforded by RAFT in

homogeneous aqueous media allows well-defined polymeric

systems to be prepared without stringent purification techniques

and under increasingly ‘‘green’’ conditions while maintaining the

ability to tailor many of the macromolecular characteristics

(molecular weight, chain topology, copolymer composition,

functionality, etc.) that affect self-assembly in solution.

We have highlighted the work in our groups and others

detailing block copolymer formation by RAFT, as well as

postpolymerization modification utilizing crosslinking and other

Soft Matter, 2008, 4, 1760–1773 | 1771

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highly efficient and orthogonal synthetic methods. Particular

attention was paid to temperature-responsive systems, but the

facile control over block copolymer structure afforded by RAFT

is useful for self-assembly in aqueous media in response to

a variety of other external stimuli. This flexibility, combined with

the capacity of RAFT to readily control block copolymer

composition, which generally dictates self assembled aggregate

morphology (micelles, vesicles, etc.), offers great potential to

prepare controlled and targeted drug delivery vehicles, biocom-

patible hydrogels, responsive polymer–protein bioconjugates,

and many other advanced materials with biological relevance.1

Indeed, RAFT polymerization and many of the postpoly-

merization modification approaches described herein have

facilitated access to ‘‘smart’’ materials with unprecedented

opportunities in biomedical, pharmaceutical, and diagnostic

areas.

Acknowledgements

CLM gratefully acknowledges financial support provided by the

Department of Energy (DE-FC26-01BC15317) and the MRSEC

program of the National Science Foundation (DMR-0213883).

BSS acknowledges the Donors of the American Chemical Society

Petroleum Research Fund (45286-G7), Oak Ridge Associated

Universities (Ralph E. Powe Junior Faculty Enhancement

Award), and the Defense Advanced Research Projects Agency

(HR0011-06-1-0032).

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