PAPER www.rsc.org/softmatter | Soft Matter

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Branched peptide actuators for enzyme responsive hydrogel particles†

Tom O. McDonald,ab Honglei Qu,abc Brian R. Saundersa and Rein V. Ulijn*abc

Received 15th October 2008, Accepted 27th January 2009

First published as an Advance Article on the web 5th March 2009

DOI: 10.1039/b818174h

We demonstrate the preparation of enzyme responsive poly(ethylene glycol) acrylamide hydrogel

microparticles (mPEGA) functionalised by solid phase synthesis with new branched peptide actuators.

Branched peptide actuators provide enhanced charge density and overcome electrostatic screening at

physiological ionic strength when compared to linear ones which do not show triggered swelling under

these conditions. Particle swelling was induced by enzymatic hydrolysis which caused a change in the

charge balance of the branched peptide actuators from zwitterionic (neutral) to cationic. Analysis of

enzymatic activity and accessibility was undertaken using fluorescence labelling and two-photon

microscopy. These experiments revealed that thermolysin could access the core of particles when linear

peptides are used, while access was restricted to the surface when using branched actuators. These

responsive mPEGA particles were then loaded with a fluorescent labeled dextran by application of

a sequential pH change. The payload could be selectively released at physiological ionic strength when

exposed to the target enzyme.

Introduction

Stimuli that have been exploited in responsive materials for use in

a biomedical context include pH,1 temperature, ionic strength

and changes in concentrations of small molecules such as

glucose. Bioresponsive materials2 are stimuli-responsive mate-

rials that change their properties in response to biochemical

recognition events, offering potential applications in biosensing,3

tissue regeneration4 and controlled release.5 The responsiveness

of these materials is determined by a biorecognition moiety that,

upon a recognition event, induces structural changes in the

material. In systems based on hydrogels there are three main

categories of molecular actuation based on changes in: (a)

crosslinking density,4,6–8 (b) electrostatic interactions9–11 or (c)

molecular conformation.12,13

There has been significant interest in exploiting enzyme cata-

lysed reactions to trigger molecular actuation in polymer

hydrogels.8,14–16 Most enzymes function under mild conditions

and some possess a high degree of selectivity. Enzymes play vital

roles in the functioning of all living systems through the tight

control of both healthy and disease specific biomolecular

processes. For example, a number of proteases (enzymes that

hydrolyse peptide bonds) have been shown to be specific markers

in many disease states including cancers17 and chronic wounds.18

Most existing enzyme responsive systems make use of cleav-

able linkers, which covalently attach a drug or drug mimic to

aManchester Materials Science Centre, University of Manchester,Grosvenor Street, Manchester, UK , M1 7HSbManchester Interdisciplinary Biocentre, The University of Manchester,131 Princess Street, Manchester, UK , M1 7DNcWestCHEM/Department of Pure & Applied Chemistry, Thomas GrahamBuilding, 295 Cathedral Street, Glasgow, UK , G1 1XL. E-mail: Rein.Ulijn@strath.ac.uk; Fax: +44 141 548 4822; Tel: +44 141 548 2110

† Electronic supplementary information (ESI) available: HPLC andLCMS traces for experiments investigating enzyme responsive swelling.See DOI: 10.1039/b818174h

1728 | Soft Matter, 2009, 5, 1728–1734

a polymer whereby enzyme action triggers release via linker

hydrolysis.19–21 We have previously introduced an alternative

approach whereby physically (rather than chemically) entrapped

payloads are released in response to enzyme cleavable peptide

actuators with rationally positioned charged groups. When

enzymatically hydrolysed, changes in electrostatic interactions of

the polymer-bound peptide fragments result in an increase in the

swelling of the polymer and payload release.10,22 Peptide actua-

tors may be designed to match target enzymes as well as payload

properties and charge.22

Stimuli-responsive polymers based on electrostatically

induced actuation are inherently highly sensitive to the ionic

strength of the solution. Indeed, our early systems were unable

to function in physiological conditions due to electrostatic

screening.22 These previous systems were based on commer-

cially available poly(ethylene glycol) acrylamide (PEGA)

macroparticles that are available in size ranging from 300 to

500 mm, for convenience in solid phase chemistry applica-

tions.23 Depending on the biomedical application smaller size

ranges may be desired (such as for injection into tissue

(<200 mm), inhalation (<100 mm) or release into circulation

(<10 mm)).24 Additionally, cell sized microparticles would

present the opportunity for rapid analysis of on-bead libraries

by using an automated cell sorter. The aims of this paper are:

(i) design of PEGA microparticles25 (mPEGA) functionalised

with peptide actuators with enhanced charge density for use

under physiological conditions (Fig. 1); (ii) comparison of the

mode of action of mPEGA functionalised with linear and

branched actuators; (iii) utilisation of these particles for trig-

gered release of a payload under physiological conditions.

As mentioned above, the inability of our previous linear

peptide actuators22 to respond at physiological ionic strength was

thought to be related to the electrostatic screening of neigh-

bouring charges. For charge induced swelling to occur the

distance between the charges must be less than the Debye length.

At physiological ionic strength (0.15 M) the Debye length is

This journal is ª The Royal Society of Chemistry 2009

Fig. 1 Schematic of the mode of action of enzyme responsive micro-

particles functionalised with branched peptide actuators. Left: molecular

structure and schematic of enzyme responsive microparticles and peptide

actuators, right: the formation of a cationic polymer upon cleavage of

a peptide by protease and the resulting charge-induced swelling of the

polymer.

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0.8 nm. Therefore, by dramatically increasing the volumetric

space occupied by the actuator within the polymer we aimed to

reduce the distance between neighbouring charges to less

than 0.8 nm. This was achieved through the incorporation of

the diamino functionalised amino acid lysine (K) to act as

a branching point from which two enzyme responsive peptide

chains were extended (Fig. 1). Each branch consists of three

parts, a diglycine (G) spacer to facilitate enzyme access, oppo-

sitely charged amino acids for electrostatically induced actua-

tion and an enzyme cleavable peptide (ECP) sequence.

Enzymatic hydrolysis of the ECP leads to release of anionic

fragments and conversion of the branched zwitterionic peptide

actuator to four cationic groups per actuator remaining cova-

lently bound to the polymer. These neighbouring cationic

groups induce swelling and an increase in the mesh size within

the polymer, which can be exploited in the triggered release of

pre-entrapped payload molecules.

Experimental

Materials

All chemicals were used as supplied and purchased from Sigma

with the exception of Fmoc-amino acids (Bachem), PEGA

macromonomers (Versamatrix), Isopar M (Multisol) and 2-(1H-

benzotriazole-1-yl)-1,1,3,3-tetra methyluronium hexa-

fluorophosphate (HBTU) (AGTC Bioproducts Ltd). The

enzymes used were thermolysin (EC 3.4.24.27), 36.5 U mg�1 and

chymotrypsin (EC 3.4.21.1), 60 U mg�1.

Inverse suspension polymerisation and polymer characterisation

A stainless steel baffleless reactor (250 ml) stirred with an anchor-

style agitator was used for the polymerisation reaction. 3.14 g

(3.4 mmol) of the PEGA800 macromonomers (2 : 1 ratio of

This journal is ª The Royal Society of Chemistry 2009

acrylamide-PEG-acrylamide to amino-PEG-acrylamide) and

0.156 g (2.2 mmol) of acrylamide were dissolved in 10 ml of

distilled water and purged for 30 min with N2 gas. 50 ml of Isopar

M (isoparaffin) was added to the reactor and was also purged for

30 min. The reactor was heated to 70 �C. After 20 min of purging

0.16 ml (1.0 mmol) of N,N,N0,N0-tetramethylethylenediamine

was added to the oil phase. 0.164 g (0.47 mmol) of Span

20 (sorbitan monolaurate) was dissolved in the oil, which was

stirred at 500 rpm for 30 s to ensure the surfactant was fully

dispersed in the oil phase. 0.070 g (0.30 mmol) of ammonium

persulfate (APS) was dissolved in the macromonomer solution,

which was added to the oil phase in the reactor and stirred at

2000 rpm for a further 30 min. The particles were washed with (3

� 50 ml) dichloromethane (DCM), (3 � 50 ml) tetrahydrofuran

(THF), (3 � 50 ml) methanol and (4 � 50 ml) distilled water.

Particle size distribution and mean particle diameter were

determined using a Malvern Mastersizer particle size analyser,

Mastersizer Microplus software version 2.18 was used to analyse

the results.

Environmental scanning electron microscopy (ESEM) images

were taken on a FEI Quanta 200 ESEM, using ESEM low vac

mode at 10.0 kV.

Solid phase peptide synthesis

Responsive microparticles with peptide actuators (linear: Fmoc-

DAAR-PEGA, branched: (Fmoc-DAARGG)2-K-PEGA) were

prepared by solid phase peptide synthesis with Fmoc protected

amino acids. 8 equivalents of the amino acid and 7.8 equivalents

of HBTU were dissolved in 2 ml of N,N-dimethylformamide

(DMF). 16 equivalents of N,N-diisopropylethylamine (DIPEA)

was added to this solution prior to its addition to mPEGA.

Coupling was carried out over 16 h and the Kaiser test26 was used

to ensure complete coupling. Deprotection was achieved using

20% piperidine in DMF for 2 h. This procedure was repeated to

build up the peptide sequence with thorough washing between

steps (5 � 5 ml methanol, 5 � 5 ml 1 : 1 methanol : DMF, 5 � 5

ml DMF). A solution of 95% trifluoroacetic acid (TFA) and 5%

water was used to remove the amino acid side chain protecting

groups.

Microscopy and determination of swelling

Swelling measurements and fluorescent images were obtained

using a Zeiss Imager A1 microscope, a Leistungselektronik mbq

52 AC power source (Jena, Germany) equipped with an HBO 50

mercury lamp and Canon Powershot G6 camera. A Zeiss filter

set 09 (excitation 450–490 nm, emission 515+ nm) was used to

visualise fluorescein isothiocyanate (FITC). For pH induced

swelling, peptide functionalised PEGA particles were immersed

in either water (CHROMASOLV plus for HPLC) or 0.01 M HCl

(pH 2.5) and images were obtained using the microscope. ImageJ

1.38x analysis software was used to determine the change in

particle diameter of a minimum of 300 particles. For enzyme

triggered swelling, individual particles were observed throughout

the timecourse and an average V/Vo was determined from at least

six representative particles, where V is the volume of the particle

at time t and Vo is the volume of the particle immediately after

exposure to the enzyme solution. Enzyme solutions were made

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up at a concentration of 1 mg ml�1 and buffer solutions were

prepared by using the appropriate amounts of sodium phosphate

dibasic heptahydrate and sodium phosphate monobasic.

Two-photon microscopy

mPEGA functionalised with either linear or branched peptide

actuators were exposed to an aqueous thermolysin solution (1 mg

ml�1) for 40 min. The particles were then washed with (5 � 1 ml)

acetonitrile (ACN) : H2O (50 : 50) containing 0.1% TFA and then

(5 � 1 ml) DMF. 6 equivalents of dansyl chloride were dissolved

in 2 ml DMF along with 10 equivalents DIPEA. This solution

was added to the particles and incubated in the dark at room

temperature for 2 h. The dansyl-labeled particles were washed

with (3 � 1 ml) DMF and (3 � 1 ml) water. Two-photon

microscopy images of the particles in water were obtained on

a Leica TCS SP2 AOBS inverted confocal using a 10� objective

lens. The confocal settings were as follows, pinhole fully open,

fully open beam expander, scan speed 400 Hz unidirectional,

format 512 � 512. Images were collected using the following

detection mirror settings: dansyl chloride 350–500 nm and

a transmitted light image using the Spectraphysics Ti-Sapphire

laser at the 750 nm laser line. The software was Leica confocal

software made by Leica Microscystems Germany. ImageJ soft-

ware was used to produce the surface plots of intensity. The

intensities at the centre, the outside (1 mm from the edge of the

particle) and the average intensity across the particle were

determined for 15 particles functionalised with either the linear

or branched peptide actuators.

Fig. 3 Characterisation of peptide functionalised responsive mPEGA. A:

HPLC quantification of Fmoc removed after each coupling step for linear

and branched peptide actuators. B: pH dependant swelling behaviour of

peptide–polymer microparticles (average taken from a minimum of 300

particles). T-test shows a difference is significant at 98%.

Entrapping payload

Particles (0.1 g) were loaded with a FITC labeled dextran (40

kDa); 2 ml of solution of FITC dextran in 0.01 M HCl (10 mg

ml�1) was added to the particles for 30 min (pH 2.5). After this

time 0.4 ml NaOH (0.05 M) was added dropwise until pH 7 was

reached. The particles were then filtered and washed (2 � 5 ml

water, 2 � 5 ml 0.18 M buffer, 2 � 5 ml methanol).

Fig. 2 A: Structure of PEGA. B: ESEM of mPEGA, inset: siz

1730 | Soft Matter, 2009, 5, 1728–1734

Release measurements

The release of entrapped dextran was determined using the

following method: loaded particles were treated in solution for 80

min; these samples were centrifuged and the supernatant was

removed. A Jasco FP – 6500 Spectrofluorometer using an exci-

tation wavelength of 490 nm was used to obtain the fluorescent

intensity at 519 nm. Treated particles were filtered and imaged

using fluorescence microscopy.

e distribution of particles obtained using the Mastersizer.

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HPLC and LCMS

HPLC experiments were undertaken on a Dionex HPLC (P680

pump, ASI-100 Automated sample injector, Nucleosil 100-5-C18

column with a UVD170U detector), using a solvent gradient of

20% ACN and 80% water to 80% ACN 20% water over 30 min

(0.1% TFA was present in both phases). Chromeleon 6.60 soft-

ware was used for analysis. All LCMS analyses were carried out

on a reverse-phase Luna C18(2), 250 � 2 mm, 5 mm column

(Phenomenex). The LCMS instrument was an Agilent 1100

Series HPLC, coupled to an Agilent 1956B mass detector. The

solvent gradient of 90% water 10% ACN to 15% water 85% ACN

over 14 min was used in all analyses; the flow rate was set at 0.5

ml min�1. Mass detection was set to analyse in SCAN mode with

electrospray ionisation.

Results and discussion

Polymer characterisation and peptide functionlisation

The first objective was to produce mPEGA by inverse suspension

polymerisation. Environmental scanning electron microscopy

(ESEM) was used to image the resulting particles (Fig. 2B),

highlighting their spherical nature. Using a Mastersizer the mean

particle diameter was found (Fig. 2B inset) to be 15 mm with

a coefficient of variation of 43%. Peptide functionalisation was

achieved through solid phase peptide synthesis using an Fmoc

protection strategy. The Kaiser test (for detection of unreacted

primary amines) and the quantification of Fmoc removed at each

Fig. 4 Enzyme responsive swelling behaviour of peptide actuator functionalis

peptide actuator, C: thermolysin in water, >: thermolysin at ionic strength

actuator, >: thermolysin at ionic strength 0.18 M, O: thermolysin at ionic

particles treated with either thermolysin or chymotrypsin at 0.18 M ionic stre

strength on total swelling of particles (after 40 min), >:thermolysin, O: no

This journal is ª The Royal Society of Chemistry 2009

deprotection step indicated high yield peptide formation (over

90% of the initial loading achieved for the final step) (Fig. 3A).

The effect of charge on the swelling of peptide modified

polymers could be determined by examining the pH dependant

swelling, dictated by amino acid side chain pKa values of 4.4 and

12.0 for aspartic acid (D) and arginine (R) respectively. There-

fore, at pH values below 4.4 these peptides have net positive

charge and electrostatic repulsion between neighbouring groups

results in an increase in swelling (Fig. 3B); at pH 2.5 there was

a 30% increase in volume for particles functionalised with linear

peptide actuator (Fmoc-DAAR-PEGA) while a 57% increase in

volume was observed with the branched sequence (branched:

(Fmoc-DAARGG)2-K-PEGA). This difference can be attrib-

uted to the double charge density present in the branched actu-

ator. pH induced swelling was later exploited to pre-load

particles with macromolecular payload at lower pH, physically

trapping them by changing back to neutral pH.

Design and responsiveness of peptide functionalised particles

Fig. 4A shows the enzyme responsive swelling of mPEGA func-

tionalised with linear peptide actuators in water upon treatment

with thermolysin (from Bacillus thermoproteolyticus rokko E.C.

3.4.24.27). Similar to our previous observations involving PEGA

macroparticles,22 at 0.18 M no swelling was observed because the

distance between charged groups in linear Fmoc-DAAR-PEGA

is greater than the Debye length (0.8 nm). mPEGA functionalised

with branched (Fmoc-DAARGG)2-K-PEGA brings cationic

ed mPEGA at pH 7. A: Swelling of particles functionalised with the linear

0.18 M. B: Swelling of particles functionalised with the branched peptide

strength 0.76 M, -: chymotrypsin in water. C: Swelling of individual

ngth (circle shows original size, scale bars are 15 mm). D: Effect of ionic

enzyme, -: chymotrypsin.

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groups closer together and doubles their density. Fig. 4B shows

an increase in volume reaching a maximum of �1.3 V/Vo after 20

min at 0.18 M confirming that this charge density is sufficient to

overcome electrostatic screening at physiological ionic strength.

Thermolysin has a relatively broad specificity preferring hydro-

phobic residues in the P10 position27 and is known to cleave AA

peptide bonds. By contrast, a-chymotrypsin from bovine

pancreas (E.C. 232.671.2), which has a preference for large

hydrophobic residues in the P1 position,28 did not give rise to

a change in swelling (visualised in Fig. 4C). Indeed, HPLC and

LCMS analysis of the solutions obtained after enzyme treatment

confirms enzymatic ECP cleavage with thermolysin (see ESI†).

Next, we evaluated the ionic strength dependence of the

branched actuators (Fig. 4D) by determining the final increase in

swelling. It was found that the maximum swelling (�1.3 V/Vo)

was observed up to an ionic strength of 0.3 M (at which the

Debye length equals 0.55 nm). Presumably, at this point the

mobile ions in solution are beginning to screen the polymer-

bound charges effectively. A minimum of �1.15 V/Vo was

reached at an ionic strength of about 0.45 M. As expected,

chymotrypsin did not initiate a response at any ionic strength

tested. In summary, functionalisation of mPEGA with the

branched peptide actuator has achieved an enzyme specific

response at physiological ionic strength.

Characterisation of enzyme action on peptide actuators

Enzymatic hydrolysis of the peptide actuators produces polymer-

bound peptide fragments with free primary amines (see Fig. 1).

The distribution of amines can be examined through labelling

with dansyl chloride, allowing for spatially resolved analysis of

enzyme action and accessibility within the particles. Two-photon

microscopy (TPM) has been demonstrated for assessing the

spatial resolution of fluorophores within polymer particles.29,30

Fig. 5 shows the distribution of dansyl-labeled amines within

mPEGA particles functionalised with either linear or branched

peptide actuators after 40 min thermolysin treatment. As seen in

Fig. 5A and B enzymatic cleavage of mPEGA functionalised with

linear peptide actuators was homogeneous throughout the

particles (on the micron scale), while branched peptide actuator

functionalised particles demonstrated enhanced fluorescence in

the outer regions of the particles. The pI of thermolysin is 4.9731

and the enzyme was therefore negatively charged at neutral pH.

Electrostatic attraction between the cationic fragments that are

left on the particle after enzymatic cleavage and the anionic

enzyme itself is expected to be stronger for branched actuators

(double the positive charge). Enzyme diffusion may therefore be

slower in particles containing branched actuators (i.e. enzymes

are held in place after enzymatic hydrolysis). Similar electrostatic

retention was observed for charged model proteins.22 The

heterogeneous distribution of enzymatic cleavage apparent for

the branched peptide actuators explains the reduced maximal

swelling observed when compared to the linear peptide actuator.

Fig. 5 Comparison of thermolysin action on both linear and branched

peptide actuator functionalised mPEGA. A: Two-photon micrographs of

representative particles labeled with dansyl moieties. B: Surface plot of

two-photon images. C: Quantification of fluorescent intensities of dansyl-

labeled, enzyme treated particles functionalised with either linear or

branched peptide actuators.

Enzyme triggered release

By exploiting the enzyme responsive swelling it was possible to

release an entrapped macromolecule in the presence of the target

enzyme. By utilising the pH responsiveness of the peptide

1732 | Soft Matter, 2009, 5, 1728–1734

actuator22 it is possible to load and entrap the payload molecules.

Here, FITC–dextran (40 kDa) was used as the payload macro-

molecule. By lowering the pH of the payload solution to pH 2.5

an increase in the swelling (Fig. 3B) and thus mesh size of the

polymer microparticles, was observed, allowing the 40 kDa

dextran to diffuse into the particles.

By returning the system to pH 7 the peptide actuator returns to

its zwitterionic uncharged form, de-swelling the particles and

physically entrapping the dextran. Fig. 6A shows a fluorescent

micrograph of mPEGA functionalised with branched peptide

actuators after washing, demonstrating the presence of FITC

labeled dextran within the particles. Fig. 6C and D shows these

particles after treatment with chymotrypsin and buffer respec-

tively (both at 0.18 M ionic strength), where the majority of the

FITC–dextran remains entrapped with some leakage apparent.

When the particles were treated with thermolysin (Fig. 6D) the

increase in swelling (and corresponding increased mesh size)

allowed the dextran to diffuse out of the particles, resulting in

a reduction of fluorescence of the particles. The amount of

FITC–dextran released into the surrounding solution was

measured by fluorescence spectroscopy (Fig. 6E); when the

particles were exposed to buffer or chymotrypsin �5 � 10�4

mmol dextran per gram of particles were released. By contrast,

This journal is ª The Royal Society of Chemistry 2009

Fig. 6 mPEGA particles functionalised with the branched peptide actuator at pH 7 (scale bars are 100 mm). A: After loading with FITC labeled 40 kDa

dextran and washing. B: After loading and 80 min exposure to thermolysin. C: After loading and 80 min treatment with chymotrypsin. D: After loading

and 80 min treatment with 0.18 M buffer. E: FITC labeled dextran released per g of particles after 80 min. (All experiments were carried out at an ionic

strength of 0.18 M.)

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treatment with thermolysin resulted in a 5.8� increase in dextran

release (Fig. 6E). Hence, the enzyme triggered increase in particle

swelling and molecular accessibility was responsible for the

greater release of the entrapped macromolecules.

Conclusions

We have demonstrated the development of a novel branched

peptide actuator that is capable of specifically responding to

enzymes at physiological ionic strength. The use of hydrogel

microparticles instead of larger macroparticles offers a faster

response compared to previous systems. This system may have

applications in a number of areas including drug delivery and

automated biosensing of ‘on-bead’ libraries using size based

separation in cell sorters.

Acknowledgements

The authors would like to thank the EPSRC for funding. The

two-photon microscope used in this study was purchased with

grants from BBSRC, Wellcome and the University of Man-

chester Strategic Fund. Special thanks goes to Robert Fernandez

for his help with the microscopy.

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