PEG-urokinase nanogels with enhanced stability and controllable bioactivity

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PEG-urokinase nanogels with enhanced stability and controllable bioactivity†

Hui Tan,a Haiqiang Jin,b Hongcheng Mei,c Lijun Zhu,a Wei Wei,a Qian Wang,a Fuxin Liang,a

Chengliang Zhang,a Jiaoli Li,a Xiaozhong Qu,*a Dihua Shangguan,c Yining Huangb and Zhenzhong Yang*a

Received 31st October 2011, Accepted 5th December 2011

DOI: 10.1039/c2sm07072c

Protein nanogels were synthesized via a one-step reaction procedure by crosslinking urokinase with

benzaldehyde bifunctionalized poly(ethylene glycol). The crosslinked architecture significantly

enhances the stability of urokinase against enzyme degradation in comparison with the core–shell

structural PEGylated proteins. Meanwhile, bioactivity of the urokinase incorporated in the nanogels

can be adjusted by varying the chain length of the corsslinking polymer. With a shorter crosslinker the

bioactivity of the uPA nanogels is seriously restricted under physiological conditions. However, the

restricted bioactivity can be completely launched by either enlarging the mesh size of the nanogel by

using longer crosslinkers, or treating the nanogels in endosomal conditions to dissociate the nanogel

structure due to the reversible conjugation chemistry.

Introduction

Polymer–protein conjugates, e.g. PEGylated therapeutic

proteins, have gained increasing interest aimed at decreasing

proteolytic degradation, prolonging circulation duration and

improving the solubility and stability.1,2 The conjugates are

normally synthesized by grafting functionalized polymers onto

the protein surface, or in situ polymerization from activated

proteins.3 The polymer moiety generates an ‘‘umbrella-like

effect’’ that can avoid the penetration of surrounding ‘‘harmful’’

bioactives like proteinase.4–6 However the steric hindrance would

significantly reduce the specific activity of the proteins.7,8

Therefore, how to balance the protective effect and the resultant

bioactivity becomes a challenge in protein conjugation. So far,

many studies focus on the structure–property relationship of the

conjugated polymers, e.g. the number, length and architecture

(linear, branched, or dendrimeric) of the polymer chains.9,10 For

example, polymers with a branched structure were found to give

a better protective effect hence longer circulation half-time to the

polymer–protein conjugates than the linear ones.11,12 On the

other hand, in comparison to the randomly modified proteins,

the synthesis of protein–polymer conjugates via site-specific

modification is considered more efficient to control the

aState Key Laboratory of Polymer Physics and Chemistry, Institute ofChemistry, Chinese Academy of Sciences, Beijing, 100190, China.E-mail: [email protected]; [email protected]; Fax: +86-10-62559373;Tel: +86-10-82619206bPeking University First Hospital, Beijing, 100034, ChinacCAS Key Laboratory of Analytical Chemistry for Living Biosystems,Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100190,China

† Electronic Supplementary Information (ESI) available: 1H NMRspectra, CD spectra and CLSM images. See DOI: 10.1039/c2sm07072c/

2644 | Soft Matter, 2012, 8, 2644–2650

bioactivity and reduce the modification level of the proteins.13

For instance, the site-specific conjugation enhances the effect of

polymer size on the shielding extent to targeting substrate.4 With

stimuli responsive polymers, the well defined protein–polymer

conjugates are able to change their properties upon external

stimulus. As such, streptavidin–PNiPAm conjugates show

characteristic of temperature dependent biotin binding.14 The

biotin binding affinity was favored below the LCST of PNiPAm,

whereas it was reduced at temperatures higher than the LCST

due to the collapse of the polymer chain around the active site

of streptavidin. This strategy has been exploited to prepare

thermo-, pH and photo-responsive protein–polymer particles

with switching activity.15,16 More recently, another approach was

proposed to control the protein protection and bioactivity via the

encapsulation of proteins inside a crosslinked polymer matrix.

The protein can be protected for more than 3 h against

proteinase. Meanwhile the single protein nanocapsule possesses

higher activity towards small substrates if the shell is non-

degradable, while for a degradable capsule layer, they react with

large substrates along with the shell degradation.17 Although the

performance is rather promising, the system suffers from a multi-

step synthesis and a lack of adaptability due to the permanent

modification of protein which may also restrict the applications

in vivo.

Herein, we report an alternative approach to simultaneously

improve the stability and control the bioactivity of proteins by

the construction of crosslinked protein–polymer nanoparticles,

i.e. protein nanogels. Polymer–protein nanoparticles have

attracted attention, because of various potential applications

including biosensing, bioseparation and drug/gene delivery.16 So

far the methods for preparing protein–polymer nanoparticles

include the conjugation of protein to polymeric or hybrid

nanoparticles,18–21 and the self-assembly of synthesized

This journal is ª The Royal Society of Chemistry 2012

http://dx.doi.org/10.1039/c2sm07072c





amphiphilic protein–polymer conjugates.22–24 In our work, the

protein nanogels were synthesized by in situ crosslinking between

protein molecules with a polymer crosslinker, i.e. poly(ethylene

glycol) (PEG) with benzaldehyde end groups. The crosslinker

reacts with the lysine residues of protein forming a dynamic

benzoic-imine linkage.25,26 The protein–polymer nanogels are

stable under physiological condition and labile at mild acidic pHs

which is promising for the intracellular delivery of proteins.

Besides, the nanogels possess two more interesting facts that may

favor the future development of protein–polymer systems.

Firstly, in comparison with the surface modified single protein,

a crosslinked structure greatly enhances the stability of the

protein nanogels against proteolytic degradation. Secondly, the

bioactivity of the protein nanogels is controllable by adjusting

the molecular weight of the crosslinking polymer (Scheme 1).

Experimental

Materials

Benzaldehyde capped polyethylene glycol (OHC-PEG-CHO)

(MW ¼ 600 Da and 2000 Da) and benzaldehyde capped methoxy

poly(ethylene glycol) (mPEG-CHO) (MW ¼ 750 Da and

2000 Da) were synthesized according to our previous work.25,26

Urokinase-type plasminogen activator (uPA) was obtained from

Livzon Pharmaceutical Group Inc., China. Chromogenic

substrate of uPA (pyroGLU-GLY-ARG-pNA.HCl, MW ¼499 Da) and the analysis kit were purchased from Chemicon

International Inc., USA. Chymotrypsin (MW � 25 kDa) was

purchased from Sigma-Aldrich, USA. Solvents and other

compounds were obtained from the Beijing Chemical Reagents

Company, China.

Synthesis of PEG–uPA conjugates

Two kinds of PEG–uPA conjugate, i.e. uPA nanogel and core–

shell structured PEGylated uPA, were synthesized. Typically,

uPA (60 mg, 1.22 mmol) and mPEG-CHO or OHC-PEG-CHO

Scheme 1 PEG–protein conjugates with a nanogel structure, i.e. PEG-uPA n

are larger than the mesh size of the nanogels from approaching, thus proh

bioactivity of proteins through changing the length/molecular weight of the cro

imine linkage in the protein nanogels results in reversible bioactivity of the p


(65.9 mmol) were dissolved in 7 mL of NaHCO3 aqueous solution

(pH� 8.0) and stirred for 24 h at room temperature. The mixture

was purified using a Sephadex G-75 gel permeation column

(column length 0.5 m, internal diameter 3 cm). The protein

containing eluate was collected and freeze-dried to gain white

powder-like product. The products were stored at �20 �C before

further characterization.

Chemical composition of the conjugates was determined by

combining methods of size exclusion chromatography (SEC) and

elemental analysis. Since core–shell uPA conjugates were gained

by the PEGylation using mPEG-CHO, the average number of

the grafted PEG chains (nPEG) on a uPAmolecule was calculated

as nPEG¼ (MWconjugate �MWprotein)/MW PEG, where MWconjugate,

MWprotein and MW PEG are the weight mean molecular weight of

the PEGylated uPA, uPA and PEG, respectively.

The reaction between uPA and OHC-PEG-CHO gave cross-

linked uPA nanogels. The chemical composition was determined

by firstly calculating the molar fractions of PEG and uPA in the

nanogel by elemental analysis through the comparison of carbon

and nitrogen content in the nanogels to that in the native protein.

Hence the average number of protein and PEG chains in

a nanogel particle (nprotein, nPEG) was calculated based on the

molecular weight data from SEC and the molar ratio of PEG to

protein (RPEG/protein) obtained from the elemental analysis, i.e.

nprotein ¼ MWconjugate/(MWprotein + RPEG/proteinMW PEG) and

nPEG ¼ nprotein � RPEG/protein.

Characterization

The molecular weight measurements were performed using size

exclusion chromatograph (SEC) equipped with a multiangle

laser light scattering and a reflection detector (MALS/RI, Wyatt

DAWN, US) on a Shodex OHpak SB-803 column (Showa

Denko, Japan) with a molecular weight limit of 1 � 106 Da

(Pullulan). Samples were dissolved in 0.1 M sodium nitrate at

5 mg mL�1 and filtered by a 0.22 mm polytetrafluoroethylene

(PTFE) disposable membrane before 50 mL of the solution was

anogels, exhibit a size exclusion effect to prevent substrates/enzymes that

ibiting the protein reaction. This strategy can be utilized to tailor the

sslinker during the nanogel synthesis. Besides, the inclusion of a benzoic-

rotein in mild acidic conditions such as in the endosome compartments.

Soft Matter, 2012, 8, 2644–2650 | 2645

injected into the column equilibrated at 37 �C and eluted with the

0.1 M sodium nitrate (containing 0.02% of sodium azide) at

a flow rate of 1 mL min�1. The dn/dc value of the sample solu-

tions was conducted using an Optilab rEX interferometric

refractometer. Elemental analysis was carried out using a Perkin-

Elmer 2400 analyzer. 1H NMR was performed on a Bruker AV

400 MHz spectrometer (Bruker Corporation, Switzerland) by

dissolving the samples in deuterated water (D2O) at required pH

adjusted using DCl and/or NaOD solution. The size and zeta

potential of the PEG-protein conjugates was measured using

a Zetasizer (Nano Series, Malvern Instruments, UK) in aqueous

solution at required pHs with a fixed concentration of 0.5 mg

mL�1. The morphology of the conjugates was observed on

a JEOL 1011 transmission electron microscope (TEM) at

200 kV. The samples were negatively stained using 0.5 wt%

phosphotungstic acid (PTA).

Biodegradation of PEG–uPA conjugates by proteinase

The biodegradation of the PEG–uPA conjugates was evaluated

by incubating the conjugates with chymotrypsin in vitro. 1 mL

solution of PEG-uPA conjugates containing an equiv of 52 IU

uPA was placed in 4 mL vials to mix with 5 IU (0.125 mg) of

chymotrypsin and the mixtures were held at 37 �C. At predicted

time points, 50 mL of the mixtures were directly injected into the

SEC to test the concentration of survived conjugates, while an

amount of the incubating solution was heated at 100 �C for 5 min

to stop the reaction. Then, the pH of the solution was adjusted to

5 using a small volume of 2 M HCl and left to stand for 4 h to

cleave the benzoic-imine bond in the conjugates and release the

uPA. The resultant solution was subsequently neutralized by

adding an equal amount of 2 M NaOH in order to analyze the

integrality of the released uPA using SDS-PAGE. The electro-

phoresis was carried out at room temperature in a vertical slab

gel apparatus. The column contained a separating gel (7.5%

polyacrylamide, 0.75 � 8 � 6 cm), overlaid with a stacking gel

(3% polyacrylamide, 0.75 � 8 � 2.5 cm). The samples were

mixed with loading buffer (containing 0.25 M Tris HCl (pH 6.8),

0.5 M DTT, 10% SDS, 50% glycerol and 0.5% bromophenol

blue) in a ratio of 1 : 1 v/v and loaded to the column together

with the protein molecular weight markers. A potential of 60 V

was applied until the dye front entered the separating gel then the

potential was increased to 110 V. Protein bands were visualized

by sensitive silver staining.

Enzymatic activity of uPA conjugates

A chromogenic substrate (pyroGLU-GLY-ARG-pNA.HCl),

which can be cleaved by uPA to generate a colored product, was

used for testing the bioactivity of uPA conjugates. uPA stan-

dards, uPA conjugate solutions (pH 7.4) and the protein conju-

gates after an acid-dissociation (pH 5.0 for 4 h) were pipetted

into 96-well plates in which the amount of uPA is equal in each

well (10 IU). The volume of the solutions in the wells was

adjusted to be 180 mL by adding 20 mL of assay buffer (pH¼ 7.4)

and sufficient amount of water. Then 20 mL of the chromogenic

substrate solution (2.5 mg mL�1) was added to the wells and the

well plates were incubated at 37 �C. Optical density of the well

plates at 405 nm (OD405) was recorded periodically using

2646 | Soft Matter, 2012, 8, 2644–2650

a microplate reader (Versamax Tunable Microplate Reader).

The experiments were conducted in triplicate.

Results and discussion

Synthesis of PEG–uPA nanogels and core–shell PEGylated uPA

Poly(ethylene glycol)–urokinase nanogels were synthesized by

conjugating the amino groups of uPA with benzaldehyde

bifunctionalized PEG (OHC-PEG-CHO). For comparison,

core–shell structural PEGylated uPAs (mPEG–uPA) were also

prepared by reacting with benzaldehyde monofunctionalized

mPEG (mPEG-CHO). The synthesis involves a one-step reaction

in aqueous media at basic pH (e.g. 7.5–8). For the synthesis of

polymer–protein conjugates, aldehyde functionalized polymers

were frequently applied for targeting the lysine residues of

proteins,27–29 however a further reduction of the produced imine

bond is normally necessary in order to strengthen the linkage.15,16

In our present work, the use of benzaldehyde capped PEG can

generate sufficiently stable PEG–protein conjugates under

physiological conditions (37 �C, pH 7.4) due to the formation of

benzoic-imine linkage.25 This is confirmed by 1H NMR from

the disappearance of the aldehdye proton peak at ca. 10 ppm in

the D2O solution of the uPA conjugates (Fig. S1, ESI†). The

PEGylation with mPEG-CHO gave core–shell structural

PEGylated uPAs (mPEG-uPA), recognized by a limited increase

of particle size compared to the native uPA (Table 1). The PEG

substitution level was evaluated based on size exclusion chro-

matography (SEC) and further confirmed by elemental analysis.

As listed in Table 1, these calculations are in good agreement,

showing that with a benzaldehyde to lysine feed ratio of

2 : 1 mol/mol, an average of 13–17 PEG chains were grafted onto

each uPAmolecule in the products which is slightly influenced by

the molecular weight of the mPEG-CHO, i.e. shorter polymer

chains result in higher substitution due to a steric favor. The

substitution level is around 50–60% to the overall 27 lysine

residues in a uPA molecule. On the contrary, the reaction of uPA

with bifunctionalized PEG (OHC-PEG-CHO) leads to larger

particles, as revealed in the TEM images (Fig. 1). The particles

contain multiple protein and PEG chains (Fig. 2a, Table 1),

indicating the formation of a crosslinked structure, i.e. nanogels.

Compared to the mPEG-uPAs, the chemical composition and

the size of the PEG–uPA nanogels are more seriously influenced

by the molecular weight (MW) of the PEG, i.e. 600 or 2000 Da

(Table 1). With the same excess amount of feed PEG, an average

of 42 and 30 PEG chains in each PEG600–uPA and PEG2000–

uPA particle are found, with which 4 and 6 uPA are crosselinked

respectively. A weak aldehyde peak is observed in the 1H NMR

spectra of PEG–uPA nanogels (Fig. S2, ESI†), indicating that

a few OHC-PEG-CHO molecules are grafted onto the proteins

with only one end. Although the calculated PEGylation level of

the uPA nanogels is ca. 10 and 5 PEG per uPA molecule, lower

than that of the mPEG-uPAs, the result can still imply the

existence of intra-crosslinking PEG chains on protein surfaces in

addition to the bridging PEG chains across different protein

molecules, since such a structure should be entropically favored.

As we previously reported, the formation of the benzoic-imine

bond is reversible upon pH variation.25,26 In this work, the drop

of pH value from neutral to mild acidic, e.g. from 7.4 to 5.0,


Table 1 Synthesis and characterization of PEG–uPA conjugates

SampleType/MWof feed PEG

Yield(%)a

MW of conjugates(kDa/Mw/Mn)

b

Ave. number of PEGand uPA chain in oneconj. particle

Hydrodynamic diameterat pH 7.4 (nm)

Zeta potential(mV)

uPA — — 49.0/1.048 — 5.2 � 0.7 85mPEG750-uPA mPEG-CHO/750 Da 87 64.2/1.074 17: 1c/15: 1d 6.3 � 0.9 0.2mPEG2000–uPA

mPEG-CHO/2k Da 89 76.6/1.070 13: 1c/12: 1d 7.3 � 0.9 3.2

PEG600–uPA OHC-PEG-CHO/600 Da 90 266.5/1.653 42: 4e 37 � 3 48PEG2000–uPA OHC-PEG-CHO/2k Da 85 334.7/1.705 30: 6e 50 � 3 60

a Calculated based on the amount of feed uPA. b Measured by SEC-MALS. c Estimated by SEC-MALS. d Estimated using elemental analysis.e Estimated based on SEC-MALS in combination with elemental analysis data.

Fig. 1 TEM images of PEG600–uPA (a) and PEG2000–uPA

nanogels (b).

resulted in the disassociation of the uPA conjugates within 3 h,

as evidenced by the decrease of particle size and MW of the

PEG–uPA nanogels down to similar values as the native uPA

(Fig. 2). Meanwhile the peak of free PEG reappears in the SEC

diagrams with increasing intensity with the acid treating time

(Fig. 2a). In addition, for the mPEG–uPA conjugates, it was

shown a recovery of the aldehyde proton peak in the 1H NMR

spectra (Fig. S1d, ESI†). Besides, circular dichroism (CD)

spectra also demonstrate a reversible transition of protein

conformation after the conjugates are dissociated (Fig. S3,

ESI†). In contrast, at physiological pH (7.4), the conjugates are

stable. No aldehyde proton was found in the 1H NMR of the

mPEG–uPAs and no obvious size change of the PEG–uPA

nanogels was observed over 7 days. Such a reversible conjugation

significantly reduces the risk of protein denaturalization, and

may endow an intracellular delivery ability of the protein–PEG

conjugates (Fig. S4, ESI†).25


Stability of the uPA conjugates against proteolytic degradation

The performance of the uPA conjugates against enzyme degra-

dation was accessed by incubating the conjugates with chymo-

trypsin (Chr, MW � 25k Da) at 37 �C, pH 7.4 for various

duration. The change of concentration of the conjugates as

a function of incubation time was monitored using SEC (Fig. 3a).

The effect of PEGylation on decreasing the enzymatic hydrolysis

rate of the protein is remarkable when compared to the native

uPA. Moreover, it is found that the uPA nanogel, e.g. the

PEG2000–uPA, is more robust to resist the proteolytic attack

than the core–shell structural uPA conjugates, i.e. mPEG2000–

uPA, within a period of 24 h. The integrality of uPA in the

conjugates after chymotrypsin incubation was further analyzed

using SDS-PAGE. The protein molecules are firstly released

from the conjugates (at pH ¼ 5.0 for 4 h) and subsequently

reduced in the presence of dithiothreitol.30 As a control sample,

the depigmentation of native uPA is observed in case the protein

was exposed to the chymotrypsin for 30 min (Fig. 3b). Mean-

while, a certain amount of uPA survives in the conjugates upon

an exposure to chymotrypsin for 2 h, demonstrating the

protective effect through PEGylation (Fig. 3c). However it is

noticed that the protective efficiency is strongly dependent on the

architecture of the conjugates, in agreement with the SEC results

(Fig. 2a). As shown in Fig. 3b and c, the Chr treated nanogels

display uPA bands with similar intensity as the untreated

samples after 2 h, although a slight enzymatic degradation can be

recognized in the lower molecular weight positions. In contrast,

the uPA band of the Chr treated mPEG–uPA conjugates is much

weaker. Note that the PEGylation levels of the mPEG750–uPA

and mPEG 2000–uPA are higher than those of the uPA nanogels

(Table 1). Considering that the molecular weight of the PEG in

the two kinds of conjugates is comparable, the results imply that

the crosslinked structure of the PEG–uPAs, i.e. the formation of

nanogel, leads to more efficient protection of the protein.

Bioactivity of the uPA conjugates to low molecular weight

substrates

A chromogenic substrate (pyroGLU-GLY-ARG-pNA.HCl,

MW ¼ 499 Da) was applied for evaluating the bioactivity of the

uPA conjugates. As shown in Fig. 4, under optimized conditions

(37 �C, pH ¼ 7.4), the enzymic reaction exhibits similar kinetics

for the two mPEG–uPAs (Fig. 4a), with 80% of the enzymic

Soft Matter, 2012, 8, 2644–2650 | 2647

Fig. 2 (a) SEC-RI diagrams of the PEG600–uPA nanogel and the core–

shell structural mPEG750–uPA in comparison with the diagrams of

native uPA, OHC-PEG600-CHO and mPEG750-CHO; and the chro-

matographic diagrams of the PEG600–uPA nanogels upon a treatment at

acidic pH (5.0) for various periods. The evolution of particle size (b) and

molecular weight (c) of the PEG600–uPA nanogels during the acidic

treatment were monitored using a zetasizer and SEC-MALS as a function

of time.

Fig. 3 SEC peak intensity (with RI detector) of uPA and different PEG–

uPA conjugates after incubating with chymotrypsin for 2–24 h (a). SDS-

PAGE investigation on the uPA released from the PEG-uPA conjugates

after the incubation with chymotrypsin (Chr) for 30 min (b) and 2 h (c).

The conjugates were dissociated by an acidic treatment in order to release

the uPA before the SDS-PAGE test.

activity with respect to the native protein during reaction for

100 h (Fig. 4c). The results imply that the polymer layer on the

mPEG–uPAs has no effect on preventing the low molecular

weight substrate from penetrating to the protein core, irre-

spective of the length of the polymer within a molecular weight

range 750–2k Da, although it has shown a moderate protection

against the binding of chymotrypsin (Fig. 3). It is reasonable

to attribute the bioactivity loss of the mPEG–uPAs (�20%

2648 | Soft Matter, 2012, 8, 2644–2650

compared to native uPA) to the denaturalization of protein

during the synthesis procedure, because a dissociation of the

conjugates did not improve the bioactivity of the released uPA in

the samples (Fig. 4c).

In comparison, the bioactivity of uPA nanogels is sensitively

related to the molecular weight of the crosslinker, i.e. the

OHC-PEG-CHO, where the nanogels exhibit huge difference in

reacting with the small substrate in case the MW of the PEG

alternated from 600 to 2k Da (Fig. 4b). While 80% of the enzymic

activity is obtained from the PEG2000–uPA, similar to that of

the mPEG–uPAs, only 11% bioactivity is displayed by the

PEG600-uPA nanogels (Fig. 4c). Besides, as shown in Fig. 4c, the

restrained bioactivity of the uPA in the PEG600–uPA nanogel

can be fully recovered by breaking the crosslinked structure.

It is commonly accepted that the lysine residue is not included

in the active site of uPA.31 Therefore the stability and bioactivity

change of the uPA conjugates would be mainly attributed to the

shielding effect from the PEG chains. Typically, the protective

effect of the polymer layer to the surface modified protein

depends on the extent of steric resistance against the approaching

of guest molecules. In the current work, for the surface

PEGylated uPA, approximately 13–17 mPEG chains, with MW

¼ 750 and 2k Da, are grafted onto a protein molecule. The zeta

potential of the PEGylated uPAs is close to neutral (Table 1),

implying that the protein surface is relatively well-masked by the

polymer chains. On the other hand, it is expected that the

shielding of PEG chains on the PEG–uPA nanogels would be

worse because the PEGylation level of the nanogels is lower. This

is demonstrated by the zeta potential of the uPA nanogels which

is very positive as the native uPA (Table 1). However beyond the


Fig. 4 Time dependent optical density at 405 nm generated from the

cleavage of pyroGLU-GLY-ARG-pNA.HCl caused by the core–shell

structural mPEG–uPA conjugates (a) and the PEG–uPA nanogels (b) at pH

7.4. Relative bioactivity of the uPA conjugates (black columns) and the

dissociated uPA conjugates (free uPA, red columns) to the native uPA (c).

The dissociated uPA conjugates were gained by an acidic treatment of uPA

conjugates at pH 5.0. And the acid treatment was also applied to the native

uPA which was used as a control sample. The acidic solutions were titrated

back to the optimized condition (pH ¼ 7.4) before the bioactivity test. No

auto-hydrolysis of the substrate was observed under the experimental

conditions. Statistics analysis: Student’s t-test, * the statistics difference is

significant, p<0.0001;NS, the statisticsdifference isnot significant (p>0.05).

prediction, the uPA nanogels exhibit much better stability

against proteinase than the mPEG–uPAs (Fig. 3). Meanwhile,

the bioactivity of the PEG–uPA nanogels shows a strong


dependence on the molecular weight of the crosslinker polymer,

which was not found on the surface PEGylated mPEG–uPAs

(Fig. 4). Furthermore, it is noticed that the nanogels conjugated

by lower molecular weight PEG are more resistive to the

surrounding molecules, which has not been experienced from the

surface modification of single proteins. Therefore it is inferred

that the protective mechanism of the PEG–uPA nanogels is

different from the shielding effect of the surface PEGylated

proteins.

By a crosslinking reaction the uPA forms a 3D network

(Scheme 1), thus the protective effect for the nanogels is mainly

influenced by the mesh size instead of the surface density of the

polymer layer as revealed from the PEGylated single-protein

conjugates.9,10,32–34 Essentially the mesh size of a polymer

network is determined by the length of the crosslinker as well as

the degree of crosslinking. Thus the uPA nanogel crosslinked

with low MW PEG, e.g. the PEG600–uPA, should exhibit low

activity to the substrate because their MW values are comparable

(600 vs. 499 Da). The size exclusion can efficiently prevent the

penetration of the substrate molecules into the network hence

limit their binding to the active sites of the protein. The low

amplitude of activity of the PEG600–uPA to the substrate might

come from the binding at the exterior surface of the nanogel

particles. Furthermore, the extension of PEG chains to a proper

length can enlarge the mesh size of the uPA nanogels which will

enable the penetration of the substrate molecules and meanwhile

can keep prohibiting the approach of larger-sized proteinase.

This is demonstrated by the property of the PEG2000–uPA.

While its bioactivity to the substrate is preserved as high as the

free proteins (Fig. 4), the PEG2000–uPA nanogel can still resist

the binding by the chymotrypsin (Fig. 3).

Conclusions

In this study, core–shell structured PEGylated uPA conjugates

and PEG-crosslinked uPA nanogels were synthesized to

demonstrate that the architecture of polymer–protein conjugates

plays an important role on the protective efficiency as well as the

bioactivity of the protein in the resultant system. The results

imply that the bioactivity of protein is controllable through the

construction of protein nanogels, by varying the molecular

weight of the crosslinker. The PEG–uPA nanogels with shorter

crosslinker exhibit a selective activity to the smaller sized

substrate of uPA while they can prevent the binding of the larger

sized chymotrypsin. Besides, the restrained bioactivity of the

PEG–uPA nanogels can be switched on in a mild acidic envi-

ronment, referring to the endosomal condition of cells, due to the

reversibility of the conjugating chemistry in the nanogel

synthesis. This might offer a possible way for the intracellular

delivery of proteins by PEGylation using the benzoic-imine

linkage.

Acknowledgements

This work is financially supported by the National Natural

Science Foundation of China (50873108, 50853001, 50733004)

and the Knowledge Innovation Program of the Chinese

Academy of Sciences (KJCX2-YW-H19).

Soft Matter, 2012, 8, 2644–2650 | 2649

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PEG-urokinase nanogels with enhanced stability and controllable bioactivity

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