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Biomaterials 31 (2010) 7555e7566

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Biomaterials

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Coreeshell hybrid nanogels for integration of optical temperature-sensing,targeted tumor cell imaging, and combined chemo-photothermal treatment

Weitai Wu a, Jing Shen a, Probal Banerjee a,b, Shuiqin Zhou a,*

aDepartment of Chemistry of College of Staten Island, and The Graduate Center, The City University of New York, Staten Island, NY 10314, USAbCSI/IBR Center for Developmental Neuroscience of College of Staten Island, The City University of New York, Staten Island, NY 10314, USA

a r t i c l e i n f o

Article history:Received 11 May 2010Accepted 22 June 2010Available online 18 July 2010

Keywords:Poly(ethylene glycol)Hybrid nanogelAgeAu bimetallic nanoparticleOptical temperature-sensingTumor cell imagingDrug delivery

* Corresponding author. Tel.: þ1 718 718 3897.E-mail address: shuiqin.zhou@csi.cuny.edu (S. Zho

0142-9612/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.biomaterials.2010.06.030

a b s t r a c t

We report a class of coreeshell structured hybrid nanogels to demonstrate the conception of integratingthe functional building blocks into a single nanoparticle system for simultaneously optical temperature-sensing, cancer cell targeting, fluorescence imaging, and combined chemo-photothermal treatment. Thehybrid nanogels were constructed by coating the AgeAu bimetallic NP core with a thermo-responsivenonlinear poly(ethylene glycol) (PEG)-based hydrogel as shell, and semi-interpenetrating the targetingligands of hyaluronic acid chains into the surface networks of gel shell. The AgeAu NP core can emitstrong visible fluorescence for imaging of mouse melanoma B16F10 cells. The reversible thermo-responsive volume phase transition of the nonlinear PEG-based gel shell cannot only modify the phys-icochemical environment of the AgeAu NP core to manipulate the fluorescence intensity for sensing theenvironmental temperature change, but also provide a high loading capacity for a model anticancer drugtemozolomide and offer a thermo-triggered drug release. The drug release can be induced by both theheat generated by external NIR irradiation and the temperature increase of local environmental media.The ability of the hybrid nanogels to combine the local specific chemotherapy with external NIR pho-tothermal treatment significantly improves the therapeutic efficacy due to a synergistic effect.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

The ability of a nanoparticle (NP) drug delivery vector that canprovide long-circulationwith adequate drug loading, illuminate thetargeted object, and intelligently sense and dose the pathologicalzones will enable major advancements in medical science anddisease treatment [1e6]. Noble metal such as Ag and Au NPspossess unique optical properties, including surface-enhanced,distance-/refractive index-dependent spectroscopic properties, andanti-photobleaching properties [7e16], thus have been extensivelyexplored for optical markers in biodiagnostic imaging [17e21].With the strong absorptions in the near-infrared (NIR) region andefficient photo-to-heat conversions (photothermal effect) [22e27],nanostructured noble metal NPs have been developed forcombined imaging and photothermal therapy [28e32]. However,these phototherapeutic systems deliver only the heat to thetumorigenic region without any drugs. It is expected that thetherapeutic efficacy will be significantly improved if drugs can be

u).

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simultaneously delivered with heat or radiation to the tumor site[33e35], which makes it possible to shorten treatment time or cutdosage.

Recently, drug delivery vectors prepared from noble metal NPswith various surface functionalizations have been explored toconjugate targeting ligands and drug molecules [5,36e42]. A keyattribute of drug delivery systems is their ability to regulate drugrelease, minimize side effects, and improve therapeutic efficacy[42,43]. Generally, these current drug delivery vectors preparedfrom the monolayer protected noble metal NPs have very low drugloading capacity. In addition, most of the capping agents on thenoble metal NPs are insensitive to local environmental change toregulate the drug release, although photoregulated release can beachieved for photosensitive drugs covalently attached to themonolayer protected metal NPs [36e38]. In order to overcomethese problems, Yoo et al. [1,44] have fabricated polymeremetalmultilayer half-shell NPs by depositing Au nanolayers onto thebiodegradable poly(lactide-co-glycolic acid) (PLGA) NPs. While thePLGA particles can serve as a drug carrier with high loadingcapacity, the Au nanolayer can absorb the NIR irradiation for pho-tothermally controlled drug release. Xia et al. [45] developeda platform based on Au nanocages covered with a monolayer ofthermo-responsive poly(N-isopropylacrylamide-co-acrylamide)

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chains. The Au nanocages can absorb the NIR light and convert it toheat, which induces the thermo-responsive polymers to collapseand thus trigger the release of the drugmolecules pre-loaded insidethe nanocages.

In this work, we aim to develop a class of coreeshell structuredmultifunctional hybrid nanogels to combine targeting, opticalsensing of environmental temperature change, fluorescenceimaging of cancer cell, adequate drug loading, and controllabledrug release into a single NP system. As illustrated in Fig. 1, thespherical hybrid nanogel particle is comprised of AgeAu bimetallicNP as core, thermo-responsive nonlinear PEG-based hydrogel asshell, and surface hyaluronic acid (HA) chains as targeting ligands.The AgeAu bimetallic NP core is designed to emit fluorescent lightfor optical sensing and cellular imaging, as well as absorb andconvert the NIR light to heat for photothermal treatment. Thenontoxic responsive nonlinear PEG-based gel shell is designed toserve as intelligent drug carriers with high drug loading capacity.The reversible swelling and shrinking of the gel shell in responseto temperature change will not only modify the physicochemicalenvironment of the embedded AgeAu NP core to manipulate theoptical properties of core for sensing on local environment, butalso change the mesh size of the gel networks to regulate the drugrelease. The surface HA chains are added to bind cluster deter-minant 44 (CD44) overexpressed on various tumors for targetingfunction [46e49]. We expect that the combination of the functionsfrom the AgeAu bimetallic NP core and responsive gel shell in thehybrid nanogels will enhance the therapeutic efficacy. Manypathological processes in various tissues and organs are accom-panied with local temperature increase by 1e5 �C [50e52]. Thisspecific temperature increase of the local pathological environ-ment (endogenous activation) can provide a biologically controlledrelease, while the NIR light can provide orthogonal externalthermal stimulus (exogenous activation) for spatiotemporalcontrol of payload release. Thus, the thermo-responsive hybridnanogels acting as drug carriers may not only provide basalchemotherapy for daily care under the endogenous activationstrategy, but also offer fast-acting dosage under exogenous acti-vation strategy, which will enhance our ability to address the

Fig. 1. Schematic illustration of multifunctional coreeshell hybrid nanogels. The AgeAu bresponsive nonlinear PEG-based gel shell cannot only manipulate the fluorescence intensityshell under the local temperature increase of targeted pathological zones or the heat genpenetrated into the surface networks of gel shell at a light penetration depth.

complexity of biological systems with remarkable spatial andtemporal resolutions.

2. Materials and methods

2.1. Materials

All chemicals were purchased from Aldrich. 2-(2-methoxyethoxy)ethyl meth-acrylate (MEO2MA, 95%), oligo(ethylene glycol)methyl ether methacrylate(MEO5MA, Mn ¼ 300 g/mol) and poly(ethylene glycol) dimethacrylate (PEGDMA,Mnz 550 g/mol) were purifiedwith neutral Al2O3. Temozolomide (TMZ), hyaluronicacid (HA, Streptococcus zooepidemicus), AgNO3, NaBH4, sodium citrate, chloroauricacid trihydrate (HAuCl4∙3H2O), L-ascorbic acid, 0.1 N HCl standard solution, 2,20-azobis(2-methylpropionamidine) dihydrochloride (AAPH), and sodium dodecylsulfate (SDS) were used as received without further purification. The water used inall experiments was of Millipore Milli-Q grade.

2.2. Synthesis of AgeAu@PEGeHA hybrid nanogels

2.2.1. Synthesis of Ag NPsCitrate-stabilized Ag NPs were first prepared by dropwise addition of fresh

NaBH4 solution (10.6 mM, 2.5 mL) to a AgNO3 solution (0.1 mM, 200 mL) in thepresence of sodium citrate (0.1 mM) under vigorous stirring. The resultant solutionwas stirred for 1 h and aged for 7 days at ambient condition to completely degradethe reducing agent of NaBH4 before use. SDS-stabilized Ag NPs were obtained byadding 3.6�10�4 mol of SDS into 200mL aqueous dispersion of citrate-stabilized AgNPs, and then aging the mixture for 10 h [53].

2.2.2. Synthesis of Ag@PEGeHA hybrid nanogelsThe Ag@PEG-HA hybrid nanogels were synthesized through precipitation

polymerization in water. In a 250 mL round-bottom flask equipped with a stirrer,a N2 gas inlet, and a condenser, 200 mL as-prepared aqueous solution of SDS-stabilized Ag NPs was heated to 30 �C, followed by addition of MEO2MA monomer(4.0 � 10‑3 mol) and PEGDMA crosslinker (5.0 � 10‑5 mol) under stirring to formhomogeneous solution. After 30 min N2 purge, the temperature was raised to 70 �Cand the polymerization was initiated by adding 1 mL of 0.105 M AAPH. The poly-merized pMEO2MA exhibits a lower critical solution temperature (LCST) in wateraround 24 �C [54e56]. Although the MEO2MA monomer is soluble in water, thepMEO2MA chains have compact conformation and are water insoluble at temper-ature above its LCST. After the polymerization was initiated at 70 �C, the hydro-phobic pMEO2MA fragments formed at the early stage of reaction would nucleateand precipitate into the hydrophobic alkyl chain regions of the SDS moleculescapping on the Ag NPs. With the proceeding of polymerization reaction, morepMEO2MA fragments were added onto the initially formed hydrophobic gel layer,leading to a continuous growth in size until the polymerization reaction was

imetallic NP (10 � 3 nm) core is NIR resonant and highly fluorescent. The thermo-of AgeAu NP core, but also trigger the release of drug molecules encapsulated in the gelerated upon NIR irradiation. HA, a known targeting ligand, can be readily semi-inter-

W. Wu et al. / Biomaterials 31 (2010) 7555e7566 7557

completed. The polymerization was allowed to proceed for 5 h. The product wascentrifuged at 20,000 rpm for 30 min (Thermo Electron Co. SORVALL� RC-6 PLUSsuperspeed centrifuge) and redispersed in 200 mL deionized water for three cycles.The resultant pMEO2MA-coated Ag NPs were used as nuclei for subsequentprecipitation polymerization to form the p(MEO2MA-co-MEO5MA) gel shell. Theshell precursors of MEO2MA and MEO5MA monomer mixture in 1:2 molar ratio,PEGDMA crosslinker (1 mol %), and SDS (3.6 � 10�4 mol) were dissolved into thecore nanogel dispersions in a 250 mL round-bottom flask equipped with a stirrer,a N2 gas inlet, and a condenser. The mixture was heated to 70 �C under a N2 purge.After 1 h, 1 mL of 0.105 M AAPH initiator was added to start the polymerization. Toincorporate the HA chains into the surface networks of hybrid nanogels, 10 mL HAsolution (3.5 mg/mL) was added dropwise to the flask after 6 h’ reaction. Thesynthesis was allowed to proceed for another 2 h to make the HA chains semi-interpenetrating into the surface networks through the hydrogen bonding betweenthe HA chains and the PEG side chains of the remained unreacted monomers. Theresulted hybrid nanogels were purified with centrifugation/redispersion inwater forthree cycles, followed by 3 days of dialysis (Spectra/Por� molecularporousmembrane tubing, cutoff 12,000e14,000) against very frequently changed water ate22 �C. Three hybrid nanogel samples were made with the feeding ofMEO5MA¼ 1.0� 10‑3 mol, 2.0� 10‑3 mol and 3.0� 10‑3 mol, respectively. They werecoded as Ag@PEGeHA hybrid nanogels.

2.2.3. Synthesis of AgeAu@PEGeHA hybrid nanogelsAgeAu bimetallic NPs inside the nanogels were prepared using the galvanic

replacement reaction [57,58]. 200 mL aqueous dispersion of Ag@PEGeHA hybridnanogels was stirred in an ice water bath for 30 min, a solution of HAuCl4 (1.0 wt %,50 mL) was then added dropwisely to the vial. The immediate color change revealedthe galvanic replacement reaction between Ag and Au(III). The solution was stirredfor another 20 min until the color was stable. After that, a solution of L-ascorbic acid(100 mM, 400 mL) was added dropwisely to allow the seed-mediated growth of Aunanoclusters. The reactionwas continued for 15 min. The final hybrid nanogels withAgeAu bimetallic NPs in the core were purified by centrifugation, decantation, anddialysis for a week against very frequently changed water ate22 �C. The resultingAgeAu@PEGeHA hybrid nanogels with the feeding of MEO5MA ¼ 3.0 � 10‑3 mol,2.0 � 10‑3 mol and 1.0 � 10‑3 mol are coded as PSG1, PSG2 and PSG3, respectively.

2.3. Incorporation of hybrid nanogels into mouse melanoma cells B16F10

Round glass coverslips were placed in wells of a 24-well plate and treated with0.1% poly-L-lysine in 100 mM phosphate buffered saline (PBS) for 40 min. Followingthe treatment, the solutionwas aspirated and thewellswerewashedwithPBS3 timeseach. Next, B16F10 cells (2� 104 cell/well) were plated on the glass coverslips at 80%confluence in DMEM containing 10% FBS and 1% penicillinestreptomycin. After 24 h,500 mL of different hybrid nanogels (3.0 mg/mL) in serum-free DMEM were respec-tively added to the marked wells. In a control well, 500 mL of serum-free DMEMwasadded. The plate was incubated at 37 �C for 2 h. The mediumwas then aspirated andfresh serum-freeDMEMwasadded toeachwell. Finally, the coverslipswith cellswereremoved from the wells and mounted onto slides for confocal microscopy study.

2.4. Drug loading and release

TMZ was loaded into the hybrid nanogels by complexation method. The pHvalue of hybrid nanogel (5 mL) was adjusted to 2.0 by using 0.1 N HCl solution. Thisdispersionwas stirred in an ice water bath for 30 min. 500 mL of TMZ solution (1 mg/mL, pH ¼ 2.0) was then added dropwisely to the vial. The hydrogen bondingcomplexation of the amide groups in the TMZ molecules with the PEG chains inhybrid nanogels caused an immediate slightly cloudy solution. After stirring over-night, the suspension was centrifuged at 5000 rpm for 30 min at 22 �C. To removefree TMZ, the precipitate was redispersed in 5 mL HCl solution of pH ¼ 2.0, andfurther purified by repeated centrifugation and washing. All the upper clear solu-tions were collected, and the concentration of free TMZ was determined by UVevisspectrometry at 328 nm. The amount of TMZ loaded into hybrid nanogels wascalculated from the decrease in TMZ concentration. The loading content is expressedas the mass of loaded drug per unit weight of dried hybrid nanogels.

The in vitro release rate of TMZ from the hybrid nanogels was evaluated by thedialysis method. A dialysis bag filled with 5 mL purified TMZ-loaded hybrid nanogeldispersion (pH ¼ 2.0) was immersed in 50 mL 0.005 M buffer solutions of pH ¼ 5.03at different temperatures. The release experiments were performed with andwithout 5 min NIR (1.5 W/cm2) irradiation at a certain time interval. The releasedTMZ outside of the dialysis bag was sampled at defined time period and assayed byUVevis absorption at 328 nm. Cumulative release is expressed as the totalpercentage of drug released through the dialysis membrane over time.

2.5. Cell viability evaluation of in vitro chemo, photothermal, and chemo-photothermal treatments

B16F10 cells (6 � 103 cell/well) were cultured in DMEM containing 10% FBS and1% penicillinestreptomycin in a 96-well plate, and exposed to free TMZ, emptyhybrid nanogels, and TMZ-loaded hybrid nanogels, respectively. The cells were

irradiated with 1.5 W/cm2 NIR light for 5 min in photothermal and chemo-photo-thermal treatments, whereas in chemotherapy alone, the cells were not exposed toNIR light. The plate was incubated at 37 �C for 24 h. The mediumwas then aspirated,and these wells were washed three times using fresh serum-free DMEM. After that,25 mL of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT)solution (5 mg/mL in PBS) were added to the wells. After incubation for 2 h, thesolution was aspirated and 100 mL of DMSO was added to each well to dissolve theformazan crystal, and the plate was sealed and incubated overnight at 37 �C withgentle mixing. Three portions of the solution obtained from each well were trans-ferred to three respective wells of a 96-well plate. Cell viability was measured usinga microplate reader at 570 nm. Positive controls contained no drug or nanogels, andnegative controls contained MTT. Parallel wells (in triplicate) also contained onlymedium (no cells) and the same concentrations of hybrid nanogels. For doublecheck, the cells were also stained with 10 mM Hoechst and cell viability was esti-mated with a Nikon fluorescence microscope fitted with a Spot digital camera, asdescribed in detail previously [6].

2.6. Characterization

The UVevis absorption spectra were obtained on a Thermo Electron Co. Heliosb UVevis Spectrometer. The photoluminescence (PL) spectra of the hybrid nanogeldispersions at different temperatures were obtained on a JOBIN YVON Co. Fluo-roMax�-3 Spectrofluorometer equipped with a Hamamatsu R928P photomultipliertube, calibrated photodiode for excitation reference correction from 200 to 980 nm,and an integration time of 1 s. To confirm all emissions, the PL spectra were alsorecorded on a VARIAN CARY Eclipse Fluorescence spectrophotometer equipped withR928 photomultiplier tubes and self-optimized light filters. The transmission elec-tron microscopy (TEM) images were taken on a FEI TECNAI transmission electronmicroscope at an accelerating voltage of 120 kV. Approximately 10 mL of the dilutedhybrid nanogel dispersionwas air-dried on a carbon-coated copper grid for the TEMmeasurements. The B16F10 cells incorporated with hybrid nanogels were imagedusing a confocal laser scanning microscopy (LEICA TCS SP2 AOBS�) equipped withan HC PL APO CS 20� 0.7 DRY len. An UV (405 nm) light was used as the light source.Dynamic light scattering (DLS) was performed on a standard light scattering spec-trometer (BI-200SM) equipped with a BI-9000 AT digital time correlator (Broo-khaven Instruments, Inc.) to measure the hydrodynamic radius (Rh) distributions. AHeeNe laser (35 mW, 633 nm) was used as the light source. All hybrid nanogeldispersions were passed through Millipore Millex-HV filters with a pore size of0.80 mm to remove dust before the DLS measurements.

3. Results and discussion

3.1. Synthesis and structure of Ag-Au@PEGeHA hybrid nanogels

The strategy to prepare the multifunctional hybrid nanogelswith nonlinear PEG hydrogel as shell and AgeAu bimetallic NP ascore involves the first synthesis of Ag NPs, followed by immobili-zation of thermo-responsive hydrogel shell on the Ag NP templates,and then a moderate growth of Au nanoclusters on the surface ofencapsulated Ag NPs. The size of highly fluorescent Ag NPs can beeasily controlled by using a dilute citrate solution [57]. Fig. 2Ashows the TEM image of spherical SDS-stabilized Ag NPs with anaverage diameter of 10 � 3 nm. The surfactant SDS might assembleto form a bilayer at the surface of Ag nanocrystals. The firstmonolayer binds with the anionic head groups pointed downtowards the surface of Ag. The exposure of the alkyl chains to theaqueous solvent is energetically unfavorable, resulting in theadsorption of a second surfactant layer with the SDS’s head groupsfacing toward the water. In turn, the bilayer would provide addi-tional stabilization and growth inhibition for Ag NPs.

The gel shell was synthesized by first coating a thin hydrophobicpMEO2MA gel layer onto Ag NP core and then using this hydro-phobic core as a seed for the subsequent formation of the morehydrophilic p(MEO2MA-co-MEO5MA) copolymer gel layer. Withthe collapsed pMEO2MA-coated Ag NPs serving as nuclei for furtherpolymerization of the p(MEO2MA-co-MEO5MA) gel layer, theformation of new polymer-only particles can be avoided. Theaddition of the p(MEO2MA-co-MEO5MA) gel layer resulted ina higher volume phase transition temperature. A proper control onthe feeding ratio of these two PEG oligomers-based monomers canlead to an LCST close to the physiological temperature [54]. HA,a copolymer of N-acetyl D-glucosamine and D-glucuronic acid, is

Fig. 2. TEM images of (A) Ag NPs, and (BeD) AgeAu@PEGeHA hybrid nanogels with different gel shell thickness: (B) PSG1, (C) PSG2 and (D) PSG3.

Fig. 3. Typical UVeviseNIR absorption spectra of Ag NPs, Ag@PEGeHA hybrid nano-gels, and AgeAu@PEGeHA hybrid nanogels (PSG1). The excitation spectrum of theluminescent PSG1 hybrid nanogels was also presented.

W. Wu et al. / Biomaterials 31 (2010) 7555e75667558

a widely used targeting macromolecule that can bind CD 44 over-expressed on various tumors [46e49]. The strong hydrogenbonding interaction between HA and PEG oligomers on the gelnetwork chains enables the formation of interpolymer complexes.Since the polymerization of PEG oligomer-based monomers isnearly completed at the sixth hour of reaction [54,56], it is expectedthat HA should locate at the light penetration depth. The semi-interpenetration of HA into the surface networks of hybrid nanogelled to an increase in the hydrodynamic size measured by DLSmethod (Fig. S1). The resultant core-shell Ag@PEGeHA hybridnanogels have a very narrow size distribution. The coating ofpolymer gel layer can be reflected in the corresponding UVevisspectra. As shown in Fig. 3, the maximum of the plasmon peak redshifted from 396 nm for Ag NPs to 408 nm for the Ag@PEGeHAhybrid nanogels.

The Ag NPs need to be engineered to increase their opticalresonances in the NIR range before they can be successfully used forphotothermal therapy. The addition of AuCl4� into the templateAg@PEGeHA hybrid nanogels can induce an immediate galvanicreplacement reaction with Ag NPs, leading to the formation ofAgeAu bimetallic NPs in the core of the hybrid nanogels. Thefeeding ratio of Au(III) to Ag(0) is controlled to be very low, so evena complete galvanic reaction would not result in a completereplacement of the Ag atoms with Au atoms. Briefly, AuCl4� oxidizesthe sacrificial Ag template to AgCl, which is highly soluble at thereaction temperature (solubility ¼ 1.92 � 10�3 g/L at 25 �C). Thestandard reduction potential of the AuCl4�/Au pair (0.99 V) is higher

than that of the AgCl/Ag pair (0.22 V). The electrons generated inthe oxidation process migrate to the surface of the Ag particles,where they reduce the AuCl4� to Au atoms. Because Au and Ag solidsshare the same face-centered cubic structure with closely matched

W. Wu et al. / Biomaterials 31 (2010) 7555e7566 7559

lattice constants (4.0786 and 4.0862 �A, respectively), the Au atomsare able to epitaxially nucleate and grow on the surface of the Agparticles [57,58]. Fig. 3 shows that the surface plasmon absorptionpeak of the Ag-Au@PEG-HA hybrid nanogels changed significantlyafter the surface modification of the Ag NP core with Au nano-clusters, whereas the shape and size of the core NPs did not showsignificant change as revealed by TEM images in Fig. 2BeD. A cor-eeshell structurewith the AgeAu NP in the core and the PEG-basedhydrogel uniformly coated on the AgeAu NP as shell was clearlyobserved. Importantly, the size of the hybrid nanogels can becontrolled by simply changing the feeding ratio of comonomers/AgNPs in synthesis. For example, the size of PSG1 (66 nm), PSG2(45 nm), and PSG3 (36 nm) correspondingly decreased when thefeeding ratio of the nonlinear PEG comonomers/Ag NPs was grad-ually decreased in synthesis. It should be noted that the size ofcolloidal drug carriers is a critical parameter to determine their fatein blood circulation since the recognition by the reticuloendothelialsystem is known to be the principal reason for the removal of manycolloidal drug carriers from the blood compartment. The sub-200 nm size is desirable for colloidal drug carrier particles to extendtheir blood circulation time [59]. The small sized hybrid nanogelspresented in this study should have advantages to extend theircirculation time and penetrate into cells.

3.2. Temperature-induced volume phase transition

Fig. 4 shows the temperature-induced volume phase transitionsof the AgeAu@PEGeHA hybrid nanogels dispersed in PBS ofpH ¼ 7.38, in terms of the change of Rh measured at a scatteringangle of q ¼ 60�. It is clear that the temperature of dispersionmedium can significantly influence the size of the hybrid nanogelsdue to the addition of thermo-responsive nonlinear PEG-based gelshell. The volume phase transitionwas detected at around 20 �C forthe pMEO2MA gel-coated AgeAu NPs. After further coating withthe relatively hydrophilic p(MEO2MA-co-MEO5MA) gel layer, thevolume phase transition of the AgeAu@PEGeHA hybrid nanogelsshifted to higher temperatures. The different thicknesses of thehydrophilic p(MEO2MA-co-MEO5MA) gel layer led to a continueschange in size but tunable slopes across the physiologicallyimportant temperature range that are found in many pathologicalprocesses in various tissues and organs (accompanied with local

Fig. 4. Temperature dependence of the average Rh values of the hybrid nanogels PSG1(-), PSG2 (C), and PSG3 (:) in PBS of pH ¼ 7.38 at a scattering angle q ¼ 60� . Theresult of the pMEO2MA-coated AgeAu NPs (;) was presented for comparison.

temperature increase by 1e5 �C) [50e52]. Therefore, the coreeshellAgeAu@PEGeHA hybrid nanogels presented in this work couldoffer two main advantages for the potential biomedical applica-tions. The small size of the hybrid nanogels with AgeAu NP coreallows the deep penetration into cell/tissue such as poorlypermeable tumors for labels and therapy, while the temperature-responsive hydrogel shell can serve as excellent drug carriers witha temperature-controllable drug releasing behavior.

3.3. Temperature-sensitive PL property of AgeAu@PEGeHA hybridnanogels

Fig. 5A shows typical PL spectra of the AgeAu@PEGeHA hybridnanogels dispersed in PBS of pH ¼ 7.38 at a low temperature of6.0 �C and a high temperature of 51.5 �C, respectively. A comparisonof the PL spectra of the Ag@PEGeHA and AgeAu@PEGeHA hybridnanogels indicates that these emissions were associated with theAg (Fig. 5 and Fig. S2). As shown in Fig. 3, the absorption spectrumof the hybrid nanogels displayed a peak near the plasmon reso-nance of Ag NPs but with a slightly broader width, suggesting thepossible presence of additional optical transitions besides plas-mons [60,61]. In contrast, the excitation spectrum adopted analmost opposite trend, exhibiting a minimum at the absorptionpeak and multiple narrow peaks superimposed on the broad trendof the excitation spectrum. These observations suggest that plas-mons do not make major contributions to the luminescence, andthe luminescence likely arises from single-electron excitationsbetween discrete energy states [61]. Scaiano et al. [10] believed thatthe emission of Ag NPs is due to small Ag cluster, predominantlyAg2 supported by the readily detectable NPs. The PL spectra of theAg in the hybrid nanogels exhibited narrow peaks superimposed ona broadband. The frequency difference between the sharp peaks isindependent of the excitation wavelength. While the intensity ofthe emissions in the 400e500 nm region was enhanced at theelevated temperature, much stronger effects were observed in thegreen light region (535e585 nm). Interestingly, a new emissioncentred at 562 nm emerged at the higher temperature.

Fig. 5B shows the more detailed temperature-sensitive PLproperty of the PSG1 hybrid nanogels in the green light region (seeFig. S3 for PSG2 and PSG3). It is very clear that the temperature-induced shrinking of the hydrogel shell could significantly enhancethe PL intensity of the Ag emissions in the AgeAu@PEGeHA hybridnanogels. To visualize the relationship between the temperature-induced volume phase transitions and PL intensity enhancement ofthe AgeAu@PEGeHA hybrid nanogels, we plotted the relative PLintensity at 545 nm as a function of temperature (Fig. 5C). It isinteresting that a linear correlation between the relative PL inten-sity and the temperatures across the physiologically importanttemperature range of 37e42 �C was observed. The comparison ofFig. 4 with Fig. 5C indicates that a conspicuous increase in fluo-rescence intensity occurred at nearly the same temperature whenthe nanogel shell began to shrink. Two factors may mainly accountfor the temperature-sensitive PL property change. One is theincrease in the local refractive index surrounding the noble metalNPs. The local optical electric field surrounding the noble metal NPscan be changed by an increase in the refractive index of the gel shelldue to the shrinkage of polymer networks, which results in anincrease in the Rayleigh scattering because of a larger refractiveindex contrast of the condensed polymer networks with thesolvent [8]. On the other hand, the different nonradiative energyloss paths, related to the reduction of the number of surface defects,provide the second scenario for the PL enhancement induced by theshrinkage of nanogels. The nonradiative energy loss paths arehighly dependent on the environmental nature around the noblemetal NPs [60]. At low temperatures, the polymer network chains

Fig. 5. (A) Typical PL profiles of the hybrid nanogels at 6.0 �C and 51.5 �C, respectively, with inset showing barely seen 545 nm and 562 nm emissions at 6.0 �C. (B) Typical PL profilesof PSG1 in the green light region, taken at 2.5 �C intervals from bottom to top except the bottom 5 curves (every 4.5 �C), from 6.0 to 51.5 �C. (C) The effect of temperature on therelative PL intensity of the three hybrid nanogels. (D) Reversible PL intensity change of the hybrid nanogels after five cycles of repeated heating (51.5 �C) and cooling (6.0 �C). Allmeasurements were made in PBS of pH ¼ 7.38. Excitation wavelength ¼ 334 nm.

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tend to expand in water. However, the bonding of the polymerchains with the noble metal NPs hinders the expansion at highlyswollen state, creating an elastic tension in the bonds at the poly-mer/metal interface, thus creating surface states that could quenchthe PL. This phenomenon is similar to the temperature-induced PLquenching of colloidal CdTe quantum dots (QDs) dispersed inwater[62]. The freezing of water induces strain in the capping shell,which can further propagate the strain to the surface of the QDs,creating surface quenching states. In contrast, at higher tempera-tures, the gel network chains are in shrunk states, which diminishthe elastic tension and consequently reduce the number of surfacetrap states acting as emission quenching centers. Importantly, thetemperature-induced PL change is fully reversible. Fig. 5D showsthe PL intensity change of the AgeAu@PEGeHA hybrid nanogelsexperiencing five cycles of heating and cooling adjustment. The PLspectra were fully reproducible after the repeated heating andcooling due to the reversible thermo-responsive volume phasetransition of the nonlinear PEG-based gel shell. The reversibleoptical property change is critical for the hybrid nanogels to senseand image the local environmental change.

3.4. Tumor cell imaging

Having demonstrated the signaling ability, the hybrid nanogelswere further evaluated as a fluorescence-labeling agent for tumorcell imaging application. Themousemelanoma B16F10 cells express

CD44 at high level [63].We expect that the AgeAu@PEGeHAhybridnanogels will effectively target and subsequently internalizethemselves into the B16F10 cells via receptor-mediated endocytosisbecause HA can bind CD44 receptor overexpressed in B16F10 cells.Fig. 6AeC shows the confocal micrographs of the B16F10 cells afterincubated with the PSG1, PSG2 and PSG3 hybrid nanogels, respec-tively. These results indicate that all HA-modified hybrid nanogelswere successfully endocytosed into the B16F10 cells. Irradiated bythe laser of 405 nm, the AgeAu NPs immobilized in the hybridnanogels produced a bright fluorescence, which retained nearly thesame PL intensity even after 20min irradiation (Fig. S5). In contrast,the fluorescence is much weaker in the B16F10 cells when stainedwith the AgeAu@PEG hybrid nanogels without HA surface modifi-cation (Fig. 6D and Fig. S6). No significant autofluorescence wasobserved under similar conditions. As the complexity of molecularinteractions governing endocytosis is revealed, the mechanisms ofendocytosis should be viewed in a broader context than simplevesicular trafficking [64e66].When the size of NPs is below100 nm,the biological pathways in cells can undergo profound changes [64].It has been reported that small sized (<100 nm) PEG-phosphati-dylethanolaminemicelles can attain deeper penetration into poorlypermeable tumors [65]. Therefore, our small sized AgeAu@PEGhybrid nanogels without surface HA ligands can still enter into theB16F10 cells, but with a lower degree of endocytosis than those HA-modified hybrid nanogels. Nevertheless, it is obvious that ourAgeAu@PEGeHAhybridnanogels serving asprobesdonot givedark

Fig. 6. Scanning confocal images of B16F10 cells incubated with AgeAu@PEGeHA hybrid nanogels of (A) PSG1, (B) PSG2, (C) PSG3, and with AgeAu@PEG hybrid nanogels withoutHA modification (D), respectively. Excitation wavelength ¼ 405 nm.

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regions in the cells and the areas where the hybrid nanogels did notpermeate are clear. It is expected that the present multifunctionalhybrid nanogels could provide complementary anatomic, func-tional, and molecular information at the cellular level needed toadvance therapy.

3.5. Drug loading and in vitro thermal- and photothermal-regulated drug release

In addition to the functions for optical temperature-sensing andcellular imaging, the designed hybrid nanogels should be alsoexcellent drug carriers benefited from the stable network structureof gel shell under typical administration conditions and the uniquephotothermal effect of the AgeAu NP core under NIR irradiation. Ahydrophilic new anticancer drug [67,68], TMZ, was selected asa model drug to demonstrate the temperature-regulated drugrelease. TMZ undergoes a chemical degradation at physiological pHto form the cytotoxic triazene, an active metabolite of dacarbazine(DTIC), which may represent a more favorable prodrug than DTIC.However, the degradation is negligible at pH � 5 [67]. We loadedthe well-dissolved TMZ into the hybrid nanogels at pH z 2. Thehydrogen bonds between the ether oxygens of the nonlinear PEGgel shell and the amide groups of TMZ molecules can reinforce thedrugepolymer interactions, resulting in a high drug loadingcapacity. A drug loading capacity of 46.5 wt%, 39.8wt% and 35.2 wt%was determined for PSG1, PSG2 and PSG3, respectively. Despite ofthe high loading capacity, the loaded drug molecules have

negligible effect on the temperature-sensitive PL property of theAgeAu NP core. The confocal images also indicate that the drug-loaded hybrid nanogels still show strong fluorescence.

The drug release from the hybrid nanogels was determined inbuffer solutions of different temperatures (Fig. 7). To avoid theevident degradation of TMZ from the long time exposure in water,the in vitro release test was made in a PBS of pH ¼ 5.03. A blankrelease experiment of free TMZ solution with an equivalentamount of drug (32.0 mg/mL) to that trapped in the hybrid nano-gels PSG3 was also performed at 41 �C (Fig. S7). The much slowerdrug release from the hybrid nanogels than from the free drugsolution indicates a sustained release of the TMZ molecules fromthe hybrid nanogels. Firstly, the temperature of the releasingmedium can trigger the drug release. The increase in temperatureinduces a gradual transition from hydrophilic to hydrophobic ofthe nonlinear PEG network chains (Fig. 4), which will not onlybreak the PEGeTMZ hydrogen bond complexes and enhance themobility of guest TMZ molecules, but also shrink down the meshsize of the nanogels so that the hydrophilic TMZ molecules areexpelled and diffuse out from the gel networks. Secondly, thethickness of the gel shell in the AgeAu@PEGeHA hybrid nanogelscan also control the drug releasing rate. The thicker the gel shell,the slower the drug releasing rate due to the longer restricteddiffusion path in the thick gel shell. Nevertheless, the tempera-ture-responsive hybrid nanogels developed in this work, beingable to switch on and off their functions when necessary underthe local temperature increase of the target pathological zone such

Fig. 7. Releasing profiles of TMZ from the PSG1 (A), PSG2 (B), and PSG3 (C) hybrid nanogels at different temperatures. The lines are based on the fitting of Empirical Peppas’s Model:Mt/MN ¼ ktn.

Fig. 8. Releasing profiles of TMZ from the PSG1 (-), PSG2 (C), and PSG3 (:) hybridnanogels immersed in buffer solutions at a constant temperature of 37 �C, irradiatedwith 1.5 W/cm2 NIR for 5 min at 0, 2, and 7 h, respectively.

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as cancer and cystic fibrosis [51e53], could be extremely impor-tant in the treatment of diseases.

To further understand the release mechanism, the results wereanalyzed on the basis of the Empirical Peppas’s model [69]:

Mt=MN ¼ ktn

where Mt and MN are the absolute cumulative amount of drugreleased to time t and infinite time, respectively; k is a structural/geometric constant, and n is a release exponent related to theintimate mechanism of release. The advantage of this approach isthat it yields a convenient measure of the constancy of release ratein the value of n. It also provides a quick way to examine thereleasing mechanism, as values of n < 0.43 or n > 0.85 for sphericaldevice indicate another releasing process in addition to the simplediffusion. The n values presented in Fig. 7 are obtained from thebest fitting of our experimental curves. While the n ¼ 0.56 for freeTMZ solution indicates a diffusion controlled transportation, thelow n values for all the hybrid nanogel samples exclude the simplediffusion controlled delivery mechanism. The n value for free TMZsolution is close to previous 0.54 found in pH ¼ 7.38 and 37 �C [6],implying that the effect of pH or temperature on the release of freeTMZ molecules can be neglected. For all hybrid nanogels, theinvestigated temperature variables did not change the drug releasemechanism, but the lower exponent n values were found at thelower temperatures. These results confirm that the release of TMZmolecules trapped in the hybrid nanogels obeys to two correlatedprocesses within the deliverymatrix. One is a chemically controlledevent related to the breakage of hydrogen bonds between the drugand the polymer chains. Another is a diffusion-controlled step.

To combine the chemotherapy with photothermal treatment,the TMZ-loaded hybrid nanogels immersed in buffer solution at37 �C were irradiated by 1.5 W/cm2 NIR light for 5 min at 0, 2, and7 h, respectively. Since the ultraviolet light may cause damage tobiological samples and can be quickly attenuated in tissue [45], weuse a NIR lampwith a filter to block ultraviolet light. Upon exposureto a NIR lamp, the AgeAu NPs embedded in the hybrid nanogels canabsorb the light and convert it into heat on a picosecond time scale,as a result of electron-phonon and phononephonon processes(photothermal effect) [70]. The heat will dissipate into thesurroundings and increase the temperature, which can induce thegel shell to shrink (Fig. 4) and thereby speed up the releasing rate ofthe pre-loaded TMZmolecules. As shown in Fig. 8, despite the shortirradiation time, more TMZ was released from the hybrid nanogelsin comparisonwith the cases without NIR irradiation (Fig. 7). Whenthe lamp is turned off, heating will immediately cease and the dropin temperature will bring the polymer chains back to its original,extended conformation, thus the drug release returns to its regular

rates. Such a NIR-triggered drug release should remarkablyenhance the ability of the hybrid nanogels for specific drug deliveryto the tumor site. While the thermo-responsive hybrid nanogelsacting as drug delivery carriers could provide basal chemotherapyfor daily care under the endogenous activation strategy, they couldalso offer fast-acting dosage under exogenous NIR activationstrategy if necessary.

3.6. Cell viability of in vitro chemo, photothermal, and chemo-photothermal treatments

A critical characteristic for future biological applications is lowcytotoxicity of a material, especially for clinical applications. Asshown in Fig. 6, no signs of morphological damage to the cells wereobserved upon treatment with hybrid nanogels for 2 h, demon-strating a minimal cytotoxicity of the hybrid nanogels. To furtherevaluate the cytotoxicity of the AgeAu@PEGeHA hybrid nanogelsand to verify whether the released TMZ was still pharmacologicallyactive, in vitro cytotoxicity tests were elaborately conducted againstB16F10 cells. In order to compare the cytotoxicity of the emptyhybrid nanogels with that of the drug-loaded ones, the studiedconcentrations were set on the base of the potential drugloading capacity of the nanogels. Fig. 9AeC shows that the emptyhybrid nanogels were non-cytotoxic to B16F10 cells after 24 h

Fig. 9. Comparison of B16F10 cell viability following photothermal, chemo, combined chemo-photothermal treatments with (A) PSG1, (B) PSG2, and (C) PSG3 as drug carriers,respectively (-: empty hybrid nanogels;C empty hybrid nanogels with 5 min initial NIR irradiation;:: TMZ-loaded hybrid nanogels;;: TMZ-loaded hybrid nanogels with 5 mininitial NIR irradiation). (D) B16F10 cell viability after treated with free AgeAu NPs (-: no NIR treatment; C: 5 min initial NIR irradiation) and free TMZ solutions (,: no NIRtreatment; B: 5 min initial NIR irradiation).

W. Wu et al. / Biomaterials 31 (2010) 7555e7566 7563

incubation in concentrations of up to 107.5 mg/mL, 125.6 mg/mLand 142.0 mg/mL for PSG1, PSG2 and PSG3, respectively. In contrast,the cell viability drastically decreased when the cells were incu-bated with TMZ-loaded hybrid nanogels for 24 h even ata concentration as low as 10.8 mg/mL,12.6 mg/mL and 14.2 mg/mL forTMZ-loaded PSG1, PSG2 and PSG3, respectively (equivalent toabout 25.8 mmol/L free TMZ in all systems). These results indicatethat the TMZ-loaded hybrid nanogels provide high anticanceractivity.

To investigate the effect of combined chemo-photothermaltreatment, the cell viability was measured when the B16F10 cellswere treated with empty and TMZ-loaded hybrid nanogels for 24 h,respectively, but adding 5 min initial NIR irradiation. Fig. 9AeCshows that the combined TMZ and photothermal treatment ismuch more cytotoxic than the chemotherapy or photothermaltreatment alone, which indicates that the therapeutic efficacy canbe significantly improved with the synergistic effect from chemo-and photothermal-therapy when the AgeAu@PEGeHA hybridnanogels are used as drug delivery carriers. Two possible reasonscan explain this synergistic effect. First, the absorption of NIR lightby the hybrid nanogels can produce local heat, which shrinks downthe gel shell network and enhance the releasing rate of TMZ drugmolecules. Second, O2 may work as an electron acceptor forphotoexcited Ag NPs [71], and the products may involve in thecytotoxicity. It has been reported that the semiconductor NPs likeTiO2 can absorb energy from light, and then transfer molecularoxygen to cytotoxic reactive oxygen species, such as hydroxyl (OH)and peroxy (HO2) radicals, superoxide anions (O2

�), hydrogen

peroxide (H2O2), and singlet oxygen (1O2) [72]. Fig. 9D shows thecytotoxicity of the free TMZ solutions at the same concentrations asthose loaded into hybrid nanogels and the free AgeAu bimetallicNPs in the absence and presence of initial 5 min NIR irradiation.Firstly, the cytotoxicity of TMZ-loaded hybrid nanogels is lowerthan that of free TMZ solution at all the studied concentrations. Thiscan be attributed to the sustained-release property of the TMZ-loaded hybrid nanogels. Considering that less than 55% of theloaded TMZ was released in 24 h under physiological conditions(Fig. 7), the slightly lower cytotoxicity of the TMZ-loaded hybridnanogels than the free TMZ solutions is understandable. Secondly,the addition of initial 5 min NIR irradiation has no effect on thecytotoxicity of the free TMZ solutions, which indicates that thephotothermal effect can only be observed in the presence ofAgeAu@PEGeHA hybrid nanogels. Thirdly, the B16F10 cells stillmaintained viability after incubated with free AgeAu NPs for 24 heven at higher dosages than those contained in the hybrid nano-gels. The addition of initial 5 min NIR irradiation on the cellsincubated with free AgeAu NPs slightly reduce the cell viability,which might be attributed to the local temperature increase afterabsorbing the NIR light. When temperature is well above thetemperature at which irreversible tissue damage occurs (40 �C), it ispossible that localized cell death can happen. Nevertheless, thetherapeutic efficacy of combined chemo-photothermal therapywith the TMZ-loaded hybrid nanogels as drug carriers was higherthan the additive therapeutic efficacy from chemo- and photo-thermal therapy alone. Fig. 10 shows a comparison of the thera-peutic efficacies from combined chemo-photothermal treatment

Fig. 10. Therapeutic efficacies of photothermal, chemo, and combined chemo-photo-thermal treatments with PSG1 (A), PSG2 (B) and PSG3 (C) as drug carriers, respectively.The efficacy of combined treatment is compared with the additive efficacy of indepen-dent chemo- and photothermal treatments using t testwith all P-values lower than 0.05.

W. Wu et al. / Biomaterials 31 (2010) 7555e75667564

(calculated by subtracting the cell viability from 100%) with theadditive therapeutic efficacies of chemo- and photothermal treat-ments, which were estimated using the relation ofTadditive ¼ 100 � (fchemo � fphotothermal) � 100, where f is the fraction

of surviving cells after each treatment [73]. When t-test is used tocompare the efficacy of combined treatment with the efficacies ofchemo- and photothermal treatments and their additive value, allP-values are lower than 0.05, indicating a significant difference.Therefore, the synergistic effect of chemo-photothermal treatmentis clearly demonstrated on the AgeAu@PEGeHA hybrid nanogels.

4. Conclusion

We have successfully developed a class of multifunctionalhybrid nanogels (<100 nm) through coating the AgeAu NPs(10 � 3 nm) with the thermo-responsive gel shell based on thenonlinear PEG oligomers for integration of optical temperature-sensing, tumor cell targeting and imaging, and thermo-/photo-thermal-regulated drug delivery. The targeting macromolecule HAcan be semi-interpenetrated into the surface networks of the gelshell at a light penetration depth through the hydrogen bondingbetween the nonlinear PEG macromonomers and HA chains. Theresulting AgeAu@PEGeHA hybrid nanogels emit strong visiblefluorescence for combined sensing on the temperature change oflocal environments and imaging on tumor cells. The hybrid nano-gels can overcome cellular barriers to enter the intracellular regionand light up the mouse melanoma B16F10 cells. The hybrid nano-gels can serve as excellent drug carriers, which cannot only providea high loading capacity for a model anticancer drug TMZ, but alsooffer a thermo-triggered release of the drug molecules in the gelnetwork. The drug molecules loaded into the hybrid nanogels canbe released more rapidly by either the temperature increase of thelocal microenvironments (endogenous activation) or the heatgenerated by NIR irradiation (exogenous activation). While theempty hybrid nanogels are nontoxic to cells, the combined chemo-thermal treatments yield higher therapeutic efficacy in comparisonwith photothermal treatment (without drug) alone, chemotherapy(without NIR irradiation) alone, and their additive efficacy, thusdemonstrating a synergistic effect. Small, stable, and nontoxichybrid nanogels with integration of multiple functionalities willoffer broad opportunities for sensing, cellular imaging, and highefficacy treatment of diseases.

Acknowledgement

We gratefully acknowledge the financial support from the USAgency for International Development under the USePakistanScience and Technology Cooperative Program (PGA-P280422) andthe National Science Foundation (CHE 0316078). We also thankPhyllis Langone at the CSI/IBR Center for Developmental Neuro-science for her help with cell culture.

Appendix

Figures with essential color discrimination. Figs. 1,3e10 in thisarticle are difficult to interpret in black and white. The full colorimages can be found in the online version, at doi:10.1016/j.biomaterials.2010.06.030.

Appendix. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.biomaterials.2010.06.030.

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