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Colloids and Surfaces B: Biointerfaces 84 (2011) 103–110

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

journa l homepage: www.e lsev ier .com/ locate /co lsur fb

he dual temperature/pH-sensitive multiphase behavior of polyN-isopropylacrylamide-co-acrylic acid) microgels for potential application inn situ gelling system

ei Xionga, Xiang Gaob, Yanbing Zhaoa,∗, Huibi Xub, Xiangliang Yanga

College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, PR ChinaSchool of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, PR China

r t i c l e i n f o

rticle history:eceived 24 September 2010eceived in revised form 8 December 2010ccepted 14 December 2010vailable online 21 December 2010

eywords:-Isopropylacrylamide

a b s t r a c t

Poly(N-isopropylacrylamide-co-acrylic acid) microgels (PNA) may be an excellent formulation for in situgelling system due to their high sensitivity and fast response rate. Four monodispersed PNA microgelswith various contents of acrylic acid (AA) were synthesized by emulsion polymerization in this paper.Their hydrodynamic diameters decreased reversibly with both decreasing pH and increasing tempera-ture. The dual temperature/pH-sensitivity was influenced by many factors such as AA content, cross-linkdensity and ion strength. In addition, high concentration PNA dispersions underwent multiple phasetransition according to different temperatures, pHs and concentrations, which were summarized in a

crylic acidicrogels

hase transitiononcentrated dispersionemperature/pH-sensitive

3D sol–gel phase diagram in this study. According to the sol–gel phase transition, 8% PNA-025 disper-sion maintained a relatively low viscosity and favorable fluidity at pH 5.0 in the temperature range of25–40 ◦C, but it rapidly increased in viscosity at pH 7.4 and gelled at 37 ◦C. This feature enabled thedual temperature/pH-sensitive microgels to overcome the troubles in syringing of temperature sensitivematerials during the injection. Apart from this, PNA could form gel well in in vitro (e.g., medium andserum) and in in vivo with low cytotoxicity. Therefore, it is promising for PNA to be applied in the in situ

gelling system.

. Introduction

Recent years, in situ gelling systems have attracted consid-rable interest for various biomedical applications [1–3]. Theyndergo a sol–gel phase transition at desired tissues or organsainly owing to three mechanisms namely pH-triggered [4,5],

emperature-dependent [6–8] and ion-activated [9]. Compared toreformed systems, the in situ gelling systems can fill any shaper defect and can be easily formulated with cells or drugs by sim-ly mixing due to their flowing nature, and do not require surgeryor placement/withdrawal [10,11]. So far, many stimuli-sensitiveopolymers have been used in this field. However, preparation ofdeal materials, which have short gelation time, proper gelationemperature and/or pH, appropriate mechanical strength, biocom-atibility, proper persistent time, convenient practical procedure

nd so on, is still strongly desired [12].

Microgels are particles made by chemically cross-linking a poly-er to form particles of a gel that are colloidal in size [13].

hey have a tunable chemical composition and physical structure

∗ Corresponding author. Tel.: +86 27 87792147.E-mail address: zhaoyb@mail.hust.edu.cn (Y. Zhao).

927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfb.2010.12.017

© 2010 Elsevier B.V. All rights reserved.

enabling control over water content, mechanical properties, andbiocompatibility [14]. Microgels can intensively change their prop-erties responsive to small stimuli such as temperature, pH, electricfield, and light [15–18]. It is well known that microgel disper-sions have lower viscosity, stronger shear-thinning capacity, higherstrengths and faster gelation response rate than corresponding lin-ear polymer solutions [19–23]. Especially, concentrated microgeldispersions exhibit a much richer phase behavior in comparisonwith linear polymers because these nanometer sized gel particlescan interact to form large networks [24–27]. Therefore, the micro-gels may be a more suitable formulation for in situ gelling systems.

In our previous work, microgels were used in the in situdrug delivery and embolization [28,29]. They were temperature-sensitive, which were generally liquid formulations at roomtemperature and able to form a semi-solid depot after injection.However, liking other polymer solutions [30–32], premature gela-tion and sticky state, which led to troubles in syringing and evenblockage of a needle during the injection of microgel dispersions

into deep anatomical sites in the body, hindered their applications.Thus the pH-sensitivity was considered to be benefit for the micro-gels to overcome these problems.

In this study, a series of dual temperature-/pH-sensitive poly(N-isopropyl-acrylamide-co-acrylic acid) (PNA) microgels were pre-

104 W. Xiong et al. / Colloids and Surfaces B: Biointerfaces 84 (2011) 103–110

Table 1F

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2

2

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eeding amounts of AA in copolymerization of PNA microgels.

Nanogel PNA-000 PNA-010

AA amounts 0 1.0 mmol

ared by emulsion polymerization with different compositionatios. Although there were some studies on the temperature/pHensitivity of PNA [33–35], their complicated phase transitionsere rarely reported. From an application point of view, however,

he proper sol–gel transition behavior of PNA was crucial for ann situ gelling system. So the dual temperature/pH-dependent mul-iphase behavior of concentrated microgel dispersions was focusedn, and a suitable formulation of microgels which in situ gellingorked well in in vitro and in in vivo was selected. Their cytotoxicity

nd biocompatiblity were also investigated.

. Materials and methods

.1. Materials

N-Isopropylacrylamide (NIPAM; Acros) and N,N′-ethylenebisacrylamide (MBA, Tianjin Kermel) were

ecrystallized from n-hexane and methanol respectively. Acryliccid (AA; Aldrich) was distilled before use. Other reagents werenalytic grade and used as received. Milli-Q ultrapure water wassed through all experiments.

Mouse embryonic fibroblast cells (NIH 3T3 cells) were obtainedrom CCTCC (China Center for Type Culture Collection). Dulbecco’s

odified Eagle’s medium (DMEM), fetal bovine serum (FBS) andew bovine serum (NBS) were purchased from Gibco. 3-(4,5-imethylthiaol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) wasurchased from Sigma–Aldrich.

.2. Preparation of PNA microgels

PNA microgels were prepared using emulsion polymeriza-ion described before [28,29]. A solution of 1.300 g (11.5 mmol)f NIPAM, 0.100 g (0.65 mmol) of MBA, 0.058 g (0.20 mmol) ofodium dodecylsulfate (SDS) and the desired amount of AA, dis-olved in 190 ml of water was prepared in a three-necked flaskquipped with a reflux condenser, a gas inlet and a thermome-er. The respective solutions were heated to 70 ◦C with magnetictirring at 300 rpm and purged with nitrogen at least 30 min toemove the dissolved oxygen. Polymerizations were initiated byhe rapid addition of 10 ml of potassium persulfate solution (KPS,.070 g, 0.26 mmol), and allowed to progress for 5 h at 70 ◦C. Theesultant PNA microgels, designated as PNA-000, PNA-010, PNA-25, PNA-050 and PNA-100 respectively according to the feedingmount of AA as shown in Table 1, were further purified by intenseialysis lasting 2 weeks with daily water exchange in order toemove residual monomers and other small molecules. After dial-sis the microgels were freeze-dried resulting in white solids forurther use.

.3. Potentiometric titration

The incorporated amount of AA monomer was obtained by titra-ion with 0.1 mol/l of KOH solution under stirring and nitrogentmosphere at 25 ◦C using the pH meter (PHS-3C, Shanghai Pre-ision Scientific Instrument Co., Ltd.) [36]. The freeze-dried PNA

icrogels were heated over phosphorus pentaoxide at 102 ◦C in a

acuum until constant weights were reached and then dispersednto 0.06 mol/l of KNO3 solution to the concentration of 0.5% (m/v).ri-distilled carbonate-free water was used as the solvent for thereparations of PNA dispersions and KOH titration. The AA amounts

PNA-025 PNA-050 PNA-100

2.5 mmol 5.0 mmol 10 mmol

were calculated by the following formulas and were also shown inTable 2.

Calculated AA amount (%)

= Feeding amount of AA (g)Total amount of comonomers (g)

× 100 (1)

Determined AA amount (%)

= KOH titrant volume (ml) × 0.1 (mol/l) × 60 (g/mol)1000 × PNA dispersion volume (ml) × 0.5%

× 100

(2)

2.4. Photon correlation spectroscopy (PCS)

The hydrodynamic diameters (Rh) of microgel particles weremeasured under various temperatures, pHs and salt conditionsby PCS (Nano-ZS 90, Malvern) equipped with a He–Ne laser(� = 633 nm). The concentration of PNA dispersions was 0.5% (w/v).At least 10 min was allowed for the samples temperature toreach equilibrium before measurement. The salt concentration wasadjusted by KNO3.

2.5. Transmission electron microscopy (TEM)

The morphologies of PNA microgels were characterized usingTEM (Tecnai G2 20, FEI, Netherlands). 0.01% (w/v) microgel dis-persions were dropped onto carbon film-coated 300-mesh TEMcopper grids for 15 min and stained with 1% (w/v) phosphotungsticacid. The copper grids were freeze-dried and air-dried at 50 ◦Crespectively over 24 h in order to investigate the morphologicdifference at various temperatures. The accelerating voltage was200 kV.

2.6. Sol–gel transition behavior of high-concentration PNAaqueous dispersions

The microgel dispersions were prepared by the mixing freeze-dried PNA microgels with 1.0 ml of citric acid/disodium hydrogenphosphate buffer solution at various pH values. After stirringovernight for swelling fully, sol–gel phase transition temperaturesof PNA microgels were studied as a function of concentration andpH by tube inverting method in the temperature range of 15–45 ◦C[37], and then mapped as phase diagrams.

2.7. Measurement of viscosity

The influence of temperature and pH on the viscosity of thehigh-concentration PNA aqueous dispersions was monitored witha rotating cylinder viscometer (Brookfield, DV-II+Pro). The rotorspeed was 20 rev/s.

2.8. Cell culture and cytotoxicity assays

Cytotoxicity of the PNA microgels was determined by MTTmethods. NIH 3T3 cells were seeded in 96-well plates at5000 cells/100 �l/well maintained in DMEM supplemented with10% heat-inactivated FBS, 100 U/ml penicillin, and 100 �g/ml strep-tomycin at 37 ◦C in a humidified incubator with 5% CO2 atmosphere

W. Xiong et al. / Colloids and Surfaces B:

Table 2Characterization of PNA microgels (n = 3).

Microgels AA amounts (%) Size (nm)a PDIa

PNA-000 0b – 146.0 ± 11.8 0.131 ± 0.024PNA-010 4.7b 4.6c 188.6 ± 12.5 0.093 ± 0.011PNA-025 10.9b 10.2c 299.7 ± 17.6 0.089 ± 0.010PNA-050 19.7b 16.7c 415.3 ± 27.1 0.155 ± 0.037PNA-100 32.9b 27.2c 811.5 ± 43.9 0.127 ± 0.016

a ◦

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2

imiaptwdhcp

3

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◦

Fd

Determined by PCS at 25 C. PNA microgels dispersed in salt-free water at.5 wt%.b Calculated AA amount using the formula (1).c Determined AA amount using the formula (2).

or one day. 100 �l of various concentrations of PNA dispersionsere then added into the culture medium. After incubation for dif-

erent periods of time, the medium was replaced by 200 �l of MTTolution (5 mg/ml) and then incubated at 37 ◦C in dark. 4 h later,TT medium was removed from each well and 200 �l DMSO was

dded, and then the mixture was stirred at room temperature. Theptical density (OD) at 490 nm was measured on a Victor3 V 1420ultilabel Counter (Perkin-Elmer, USA).

.9. In vivo gel formation

Wistar rats (male, 220–250 g, 10 weeks) were used to exam-ne the in vivo gel formation behavior and biocompatibility of PNA

icrogels. The animals were housed individually in plastic cagesn a controlled environment with free access to food and water. Allnimal experiments were performed in accordance with the Peo-le’s Republic of China national standard (GB/T 16886.6–1997). Aotal of 0.5 ml of PNA dispersion at pH 5.0 and room temperatureas injected subcutaneously using a 5 cm3 syringe with a 23 G nee-le into the dorsum of a rat anesthetized with 10% (w/v) chloralydrate. At a predetermined time, the animals were sacrificed byervical dislocation. The injection site was carefully cut open andhotographed.

. Result and discussion

.1. Preparation and characterization of PNA microgels

The dual temperature/pH-sensitive copolymer microgels con-isted of cross-linked NIPAM and AA had a spherical structurend exhibited a low polydispersity at swollen state as shown in

ig. 1. TEM images of PNA microgels: (A) micro-morphology of PNA microgels treated wrying.

Biointerfaces 84 (2011) 103–110 105

Fig. 1A and Table 2. It seemed that the PNA particles showed anobvious core–shell structure with a black core and surroundingshadows, indicating that the microgels might have an inhomo-geneous cross-link density, which gradually decreased from thecenter to the periphery. It was in agreement with the observa-tions of other groups [38–40]. It is well known that PNIPAM is atypical temperature-sensitive polymer. The samples for the TEMwere attained by freeze-drying and vacuum drying at 50 ◦C respec-tively. Therefore, the freeze-dried PNA microgels were swollen withlarger diameter (approximate 267 nm) (Fig. 1A) and collapsed tothe irregular shape and smaller size (approximate 166 nm) by vac-uum drying above its lower critical solution temperature (LCST)(Fig. 1B).

AA was used as co-monomer to confer charged functionalitiesto the microgels and therefore influenced their phase transitionbehaviors. The incorporated amounts of AA in the PNA microgelswere determined by titration and shown in Table 2. Consideringthe calculated and determined AA content, the incorporation effi-ciency of AA residues within microgels decreased with the increasein the feeding amount of AA monomers, but at least 80% of the ini-tial AA was copolymerized in the microgels. It might be resultedfrom a higher extent of generation of PAA homopolymers whichwere then entirely or almost entirely removed by dialysis duringcopolymerization of high-concentration AA [41].

3.2. Temperature/pH-sensitivity of PNA microgels

Due to introduction of ionizable monomer units into the PNI-PAM polymer network, the PNA microgels could respond to bothtemperature and pH changes in aqueous solution. Particularly, withrespect to parameters such as ionic strength and pH, their sensi-tivity was apparently enhanced compared to PNA-000s. As shownin Fig. 2, the hydrodynamic diameters of the copolymer micro-gels decreased with increasing temperature, but increased withincreasing pH value. The temperature sensitivity was originatedfrom the decreases of the hydrogen bonding force between waterand polymer segments when elevating the temperature [42–44].Some experiments suggested that, as to highly charged PNA micro-

gels, a second de-swelling process appeared at about 60 C [33]. Inthis study, however, the microgels were studied just in the tem-perature range of 20–45 ◦C for biological application, so no twostep transition process was evident in the monitored temperaturewindow.

ith freeze-drying; (B) micro-morphology of PNA microgels treated with vacuum

106 W. Xiong et al. / Colloids and Surfaces B: Biointerfaces 84 (2011) 103–110

Fig. 2. The hydrodynamic diameters of PNA microgels particles as a function oftemperature and pH. (A) 0.5 wt% of salt-free aqueous dispersions of PNA microgelsa2s

towtmtinclwPgiic

eimNcpsoten

dispersions resulted in the formation of colloidal glass [56]. With

t various temperatures; (B) 0.5 wt% of PNA microgels dispersions at various pHs at5 ◦C. 0.1 mol/l of HCl or KOH solution was used to adjust the pH value of the testingamples.

The pH-sensitivity of PNA microgels was due to deprotona-ion/protonation of AA, which was influenced by the fluctuationf external pH value [45–46]. The enhanced ionization of net-ork charge in the pH-increasing solution was able to increase

he Coulomb repulsions among the polymer chains and accom-odate an additional osmotic contribution to the swelling. Since

he increase of solution pH, PNA-000 remained nearly unchangedn size, but copolymer microgels significantly expanded until theetwork charge ionized completely. Particularly, due to its high AAontent, the PNA-100 microgels swelled to a great extent, whiched to its size was undetectable by PCS because its refractive index

as very close to the refractive index of water. Besides, the size ofNA-050 below pH 4 was also not shown in Fig. 2B due to aggre-ation and precipitation of the microgels at low pH. It might benduced by the high amount of inter-particle hydrogen bondingnteractions between the amide group and the neutral form of thearboxylic moiety.

The temperature/pH-sensitivity of PNA microgels was influ-nced by many factors such as AA content, cross-link density andon strength. The series of PNA microgels synthesized by copoly-

erization of various amounts of AA with constant amount ofIPAM and MBA had an increasing AA content and a decreasingross-link density. Thus with the increasing amount of incor-orated AA and the decreasing cross-link density, the size,welling/de-swelling ratio, LCST and temperature/pH-sensitivity

f PNA microgels all increased, which were in agreement withhe results of previous reports [33,47–50]. These effects could bexplained as being due to both an increased hydrophilic microgelsetworks resulting from AA content rise and a decreased polymer

Fig. 3. The hydrodynamic diameters of PNA microgels particles treated with variousconcentrations of KNO3 at 25 ◦C at pH 7.0.

elasticity caused by cross-link density decline. Especially in the sizeof PNA-100, it exhibited a continuous linear decay with tempera-ture and did not seem to be compatible with the existence of acritical temperature.

In general, salt could reduce the repulsive electrostatic forceswithin the polymer matrix due to decreasing the Debye screen-ing length and tended to disrupt or distort the water molecules inhydrophobic hydration shell around the particles [51] as well asreduce the osmotic pressure caused by mobile counterions [52,53].Therefore, the increase in ionic strength led to collapse of PNAmicrogels, as shown in Fig. 3. However, when the ion strength wassufficient to suppress electrostatic effects, the size of PNA micro-gels were independent with salt concentration and decreased toa constant consequently. The critical salt concentration estimatedfrom the figure was about 0.05 mol/l.

3.3. Multiphase transition of high-concentration PNA microgelsdispersions

Fig. 4 showed the phase transitions of PNA microgels in salt-free aqueous dispersions. The concentrated dispersions exhibitedrich phase behavior which was easily observable from their appear-ances. At low temperature the microgel spheres were fully swollenand the PNA dispersions existed as a glassy phase (phase I). Uponincreasing the temperature, the colloidal glass became a homo-geneous translucent liquid (phase II). When the temperature wasraised further to above the LCST of PNA, the microgel particlesshrunk and the dispersions became opaque (phase III). However,the cloud dispersions remained colloidally stable and no macro-scopic phase separation was observed even preserved at constanttemperature for a long time, unless temperature increased. Thecloud suspensions subsequently immobilized at higher tempera-ture, became a white, semisolid shrunken gel (phase IV). Once thegel was formed, it did not dissolve or change its water content onadding additional water. With further increased temperature, themicrogels further shrunk and expelled water exhibited apparentphase separation and precipitated on the bottle wall (phase V).

This phase transition process of concentrated PNA dispersionswas similar to the previous results of other PNIPAM-based gelowing to that they were all controlled by the interaction betweenparticles [27,28,54,55]. At low temperature, the significant amountof hydrogen bonds and van der Waals attraction in concentrated

increasing temperature, the inter-particle potential weakened andthe colloidal glass started to flow above a critical temperaturewhich was defined as the gelation temperature (GT). Above theLCST, the sharp increase in turbidity designated the dispersions

W. Xiong et al. / Colloids and Surfaces B: Biointerfaces 84 (2011) 103–110 107

ueous

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absWPiPatTiaaTttp

3

psBdpds

w

Fig. 4. The multiphase transition behavior in salt-free aq

ntered into a two-phase domain where the phase separatingt high temperature (defined as the separation temperature, ST)as in equilibrium with clear solution. The collapsed microgels,hich formed particles were so small, had a tendency to formydrophobic aggregates [26]. Then so much of the new phase had

ormed where the particles interacted strongly, forming a gel-likeystem.

Although the phase transition of microgels was essentiallyscribed to the inter-particle potential, it was apparently affectedy many factors. For neutral microgels the potential mainly con-isted of a short-range repulsion and a longer-ranged van der

aals-like attraction, which was not sensitive in the former and ledNIPAM microgels in good agreement with the hard sphere modeln a certain concentration range [55,57]. So the phase behavior ofNIPAM microgels was apparently mainly dominated by their sizesnd distributions. But for the PNA microgels, the additional elec-rostatic effects brought by the network charge cannot be ignored.hus, pH, ion strength and AA content had to be considered, lead-ng to a different phase behavior. For example, PNA aggregated soonnd precipitated to form a phase separation in the buffer saline athigh temperature, without the formation of a white gel phase.

hus considering the dual temperature-/pH-sensitivity and poten-ial application of PNA microgels in the in situ gelling system, theemperature/pH-induced sol–gel phase transition of microgel dis-ersions had to be investigated.

.4. Sol–gel transition phase diagrams

The sol–gel phase transition diagrams of concentrated PNA dis-ersions were determined using tube inverting method. Fig. 5howed the GT with different concentrations at various pH values.ecause no immobile shrunken gel was observed in buffer salineispersions of PNA at high temperature, the GT surface in the 3D

hase diagrams was the unique sol–gel interface, above which theispersions were in sol states, but below which they were in geltates.

As shown in the phase diagrams, the GT increased reversiblyith increasing pH, microgels concentration and AA content when

dispersion of 10% PNA-010 as a function of temperature.

the pH was below 7. The GT of PNA-025 and PNA-050 significantlyenhanced at high pH range, even beyond the experimental temper-ature range. Especially of PNA-100, the GT rose to a much higherlevel, leading the dispersion to maintain colloidal glass under thewhole experimental conditions. So its phase diagram was not givenin Fig. 5. Although compared with neutral PNIPAM microgels, phasetransition behavior of PNA was more complicated, the GT wasessentially controlled by the inter-particle potential, which was theconsequence of a competition between attractive interactions andelectrostatic repulsion. At low pH, net charges in microgels werevery little, and the size-dependent attraction played a major role inmicrogels phase transition, similar as the hard sphere model [57].Therefore, the GT rise with microgel particles size, which increasedwith increase of pH and AA. However, at high pH, where electro-static repulsion could not be ignored, the inter-particle potentialmight decline, leading to a slight decrease of the GT of PNA-010 atpH 8, especially in high concentration.

At low pH, the dilute dispersion of microgels was not able toform colloidal glass phase at an even temperature below 15 ◦C, indi-cating the existence of critical gel concentration (CGC). The CGC ofPNA dispersions in buffer saline was much higher than that of PNI-PAM dispersions [54,55], which may be caused by the electrostaticeffect. It was seen in the phase diagrams that the CGC decreasedboth with increasing pH and AA content, which was also attributedto the size-dependent attraction.

3.5. In vitro and in vivo gel formation of PNA microgel

According to the PNA phase diagrams, suitable formulas of dis-persions can be chosen as the dual temperature/pH-sensitive in situgelling materials for the drug delivery and embolization. As shownin Fig. 6A, at room temperature, 8% PNA-025 dispersion lay in thesol domain at pH 5.0 and maintained the state as the temperature

rising to 37 ◦C, but became gel at pH 7.4 and 37 ◦C. This predic-tion was confirmed by experiment, which was shown in Fig. 6Bthat, when dropped respectively into pH 5.0 and 7.4 buffer salineat 37 ◦C, the 8% PNA-025 dispersion (pH 5.0) dispersed in the for-mer and immediately diffused, but formed a gel and floated in the

108 W. Xiong et al. / Colloids and Surfaces B: Biointerfaces 84 (2011) 103–110

Fc

lng

firwmn7mrd

in vivo. Although it was found from MTT assay that NIH 3T3 cells

ig. 5. The gelation temperatures of various microgels dispersions with differentoncentrations at various pH values: (A) PNA-010; (B) PNA-025; (C) PNA-050.

atter. Moreover, it can also form gel well in cell culture media andew bovine serum (NBS), promising for application in the in situelling system.

Fig. 6C showed the viscosity of 8% PNA-025 dispersion as aunction of temperature and pH. The PNA dispersion decreasedn viscosity at pH 5.0 in the temperature range of 20–40 ◦C, butemained a relatively high viscosity at pH 7.4. So, it is expected thathen injected through a catheter into the body, the 8% PNA-025icrogel dispersion (pH 5.0) would maintain the flowing state and

ot increase in viscosity, but in situ gel only in contact with a pH

.4 environments after entering the body. This feature enabled theicrogel dispersions to overcome the temperature sensitive mate-

ials’ troubles of premature gelation and even blockage in a needleuring the injection.

Fig. 6. (A) Sol–gel transition of 8% PNA-025 dispersion as a function of temperatureand pH. (B) Gelation of 8% PNA dispersion at 37 ◦C induced by injection into pH 5.0(I) and pH 7.4 (II) buffer solutions, DMEM cell culture media (III) and NBS (IV). (C)The influence of temperature and pH on viscosity of 8% PNA-025 dispersion.

In vivo gel formation of 8% PNA-025 was tested in rat modelthrough subcutaneous injection. The microgels formed a gel imme-diately after injection (Fig. 7B). The formed gel was spherical inappearance (Fig. 7C) and maintained shape at the injection sitefor the full experimental observation period. All rats were healthythroughout the experiments, associated with the clean implantedsites and no obvious injuries to the surrounding tissues, indicatingthat the microgels were biocompatible.

3.6. Cytotoxicity of PNA microgel

As shown in Fig. 8, the PNA-025 microgels did not affect themetabolic activity when added in the range of 0–50 mg/ml to NIH3T3 cell lines, showing very low cytotoxicity. Increasing incuba-tion time could slightly influence the proliferation of NIH 3T3 cells,but there were no significant statistical difference. The toxicity ofPNA-025 is one of the most important concerns for applications

grew normally even exposing to the high concentration, there werecertain limitations of in vitro cytotoxicity as a predictor of toxic-ity in vivo. Therefore, the toxicity in animal experiments would beinvestigated in future.

W. Xiong et al. / Colloids and Surfaces B:

Fig. 7. Subcutaneous injection of 8% PNA-025 (A), formed gel (B) and apparentmorphology of the gel after 3 days (C).

Fue

4

rdi

[

[[[[

[[[[

[[[[[[[

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ig. 8. Cytotoxicity of PNA-025 microgels. The cells treated without PNA-025 weresed as control, where * represented statistically difference (P < 0.05). Results werexpressed as mean ± SD (n = 3).

. Conclusions

Microgels are excellent intelligent responsive materials, butarely used in the in situ gelling system. Four PNA microgels withifferent contents of AA were prepared by emulsion polymerization

n this paper. Their dual temperature/pH-sensitivity was investi-

[

[

[

Biointerfaces 84 (2011) 103–110 109

gated, and the phase transition of their concentrated dispersions atvarious temperatures, pHs and concentrations were focussed on.The PNA microgel dispersions successively underwent the statesof colloidal glass, clear sol, cloudy sol, semisolid shrunken gel, andphase separation in salt-free water with increasing of temperature,but exhibiting a slightly different phase behavior in buffer saline.The GT of PNA dispersions increased reversibly with increasing pH,concentration and AA content, and the CGC of PNA decreased bothwith increasing pH and AA content, which were all attributed tothe size-dependent attraction.

According to the PNA phase diagrams, suitable formulas of dis-persions can be chosen as the dual temperature/pH-sensitive in situgelling materials. The 8% PNA-025 dispersion maintained a rela-tively low viscosity at pH 5.0 in the temperature range of 25–40 ◦C,but rapidly increased in viscosity at pH 7.4 and gelled at 37 ◦C. Thisfeature enabled the dual temperature/pH-sensitive microgels toovercome the defects in syringing of temperature sensitive mate-rials during the injection. PNA can also form gel well in vitro (e.g.,media and serum) and in vivo, and show low cytotoxicity. Therefore,it is promising for the applications in in situ gelling system.

Acknowledgements

This work was financially supported by the MOST 973 Program(grant no. 2007CB935800), National High Technology Researchand Development Program of China (2006AA03Z332), and Nat-ural Science Foundation of China (NSFC, 50703014). In addition,acknowledgment was also given to the Analysis and Test Center ofHUST for the TEM measurement.

References

[1] A. Hatefi, B. Amsden, J. Control. Release 80 (2002) 9.[2] C.B. Packhaeuser, J. Schnieders, C.G. Oster, T. Kissel, Eur. J. Pharm. Biopharm. 58

(2004) 445.[3] S.R. Van Tomme, G. Storm, W.E. Hennink, Int. J. Pharm. 355 (2008) 1.[4] B. Srividya, R.M. Cardoza, P.D. Amin, J. Control. Release 73 (2001) 205.[5] Y.L. Chiu, S.C. Chen, C.J. Su, C.W. Hsiao, Y.M. Chen, H.L. Chen, H.W. Sung, Bioma-

terials 30 (2009) 4877.[6] B. Jeong, S.W. Kim, Y.H. Bae, Adv. Drug Deliv. Rev. 54 (2002) 37.[7] E. Ruel-Gariépy, J.C. Leroux, Eur. J. Pharm. Biopharm. 58 (2004) 409.[8] L. Yu, J. Ding, Chem. Soc. Rev. 37 (2008) 1473.[9] H. Gupta, T. Velpandian, S. Jain, J. Drug Target 18 (2010) 499.10] Y. Liang, Y. Qiao, S. Guo, L. Wang, C. Xie, Y. Zhai, L. Deng, A. Dong, Soft Matter 6

(2010) 1915.11] L.S. Wang, J.E. Chung, P.P. Chan, M. Kurisawa, Biomaterials 31 (2010) 1148.12] C. He, S.W. Kim, D.S. Lee, J. Control. Release 127 (2008) 189.13] R.J. DeVolder, H.J. Kong, Biomaterials 31 (2010) 6494.14] J.K. Oh, D.J. Siegwart, H.I. Lee, G. Sherwood, L. Peteanu, J.O. Hollinger, K. Kataoka,

K. Matyjaszewski, J. Am. Chem. Soc. 129 (2007) 5939.15] N.A. Peppas, J.Z. Hilt, A. Khademhosseini, R. Langer, Adv. Mater. 18 (2006) 1345.16] M. Ballauff, Y. Lu, Polymer 48 (2007) 1815.17] S. Bhattacharya, F. Eckert, V. Boyko, A. Pich, Small 3 (2007) 650.18] A. Garcia, M. Marquez, T. Cai, R. Rosario, Z. Hu, D. Gust, M. Hayes, S.A. Vail, C.D.

Park, Langmuir 23 (2007) 224.19] Y. Hirokawa, T. Tanaka, Chem. Phys. 81 (1984) 6379.20] W. Funke, Br. Polym. J. 21 (1989) 107.21] B.R. Saunders, B. Vincent, Adv. Colloid Interface Sci. 80 (1999) 1.22] Z. Hu, X. Lu, J. Gao, C. Wang, Adv. Mater. 12 (2000) 1173.23] M. Stieger, W. Richtering, Macromolecules 36 (2003) 8811.24] P.S. Mohanty, W. Richtering, J. Phys. Chem. B 112 (2008) 14692.25] G. Romeo, A. Fernandez-Nieves, H.M. Wyss, D. Acierno, D.A. Weitz, Adv. Mater.

22 (2010) 3441.26] C.K. Han, Y.H. Bae, Polymer 39 (1998) 2809.27] Z.B. Hu, X.H. Xia, Adv. Mater. 16 (2004) 305.28] Q. Wang, Y. Zhao, Y. Yang, H. Xu, X. Yang, Colloid Polym. Sci. 285 (2007) 515.29] Q. Wang, H. Xu, X. Yang, Y. Yang, Int. J. Pharm. 361 (2008) 189.30] J.M. Suh, S.J. Bae, B. Jeong, Adv. Mater. 17 (2005) 118.31] Y. Yeo, C.B. Highley, E. Bellas, T. Ito, R. Marini, R. Langer, D.S. Kohane, Biomate-

rials 27 (2006) 4698.32] W.S. Shim, J.H. Kim, H. Park, K. Kim, I. Chan Kwon, D.S. Lee, Biomaterials 27

(2006) 5178.33] K. Kratz, T. Hellweg, W. Eimer, Colloids Surf. A: Physicochem. Eng. Asp. 170

(2000) 137.34] D. Huo, Y. Li, Q. Qian, T. Kobayashi, Colloids Surf. B: Biointerf. 50 (2006) 36.

1 ces B:

[

[

[[

[

[

[[[[

[

[

[

[[[

[

[[53] A. Fernandez-Nieves, A. Fernandez-Barbero, F.J. de las Nieves, J. Chem. Phys.

10 W. Xiong et al. / Colloids and Surfa

35] D. Teng, J. Hou, X. Zhang, X. Wang, Z. Wang, C. Li, J. Colloid Interface Sci. 322(2008) 333.

36] S. Ito, K. Ogawa, H. Suzuki, B. Wang, R. Yoshida, E. Kokufuta, Langmuir 15 (1999)4289.

37] A. Ghosh, J. Dey, Langmuir 25 (2009) 8466.38] A. Fernández-Barbero, A. Fernández-Nieves, I. Grillo, E. López-Cabarcos, Phys.

Rev. E: Stat. Nonlin. Soft Matter Phys. 66 (2002) 051803.39] T.G. Mason, M.Y. Lin, Phys. Rev. E: Stat. Nonlin. Soft Matter Phys. 71 (2005)

040801.40] A. Fernández-Nieves, F.J. de las Nieves, A. Fernández-Barbero, J. Chem. Phys.

120 (2004) 374.41] H.C. Chiu, Y.F. Lin, Y.H. Hsu, Biomaterials 23 (2002) 1103.

42] H.G. Schild, Prog. Polym. Sci. 17 (1992) 163.43] C. Wu, X.H. Wang, Phys. Rev. Lett. 80 (1998) 4092.44] F.A. Plamper, A. Schmalz, M. Ballauff, A.H. Müller, J. Am. Chem. Soc. 129 (2007)

14538.45] T. Tanaka, D. Fillmore, S.T. Sun, I. Nishio, G. Swislow, A. Shah, Phys. Rev. Lett. 45

(1980) 1636.

[[[[

Biointerfaces 84 (2011) 103–110

46] A. Fernandez-Nieves, A. Fernandez-Barbero, B. Vincent, F.J. de las Nieves,Macromolecules 33 (2000) 2114.

47] G.M. Eichenbaum, P.F. Kiser, A.V. Dobrynin, S.A. Simon, D. Needham, Macro-molecules 32 (1999) 4867.

48] H. Senff, W. Richtering, Colloid Polym. Sci. 278 (2000) 830.49] T. Hoare, R. Pelton, Macromolecules 37 (2004) 2544.50] M. Karg, I. Pastoriza-Santos, B. Rodriguez-González, R. von Klitzing, S. Wellert,

T. Hellweg, Langmuir 24 (2008) 6300.51] M. Annaka, K. Motokawa, S. Sasaki, T. Nakahira, H. Kawasaki, H. Maeda, Y. Amo,

Y. Tominaga, J. Chem. Phys. 113 (2000) 5980.52] I. Ohmine, T. Tanaka, J. Chem. Phys. 77 (1982) 5725.

115 (2001) 7644.54] H. Senff, W. Richtering, J. Chem. Phys. 111 (1999) 1705.55] J. Wu, B. Zhou, Z. Hu, Phys. Rev. Lett. 90 (2003) 048304.56] D. Rosenbaum, P.C. Zamora, C.F. Zukoski, Phys. Rev. Lett. 76 (1996) 150.57] C.L.A. Berli, D. Quemada, Langmuir 16 (2000) 7968.