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High-Strength Hydrogels with Integrated Functions of H-bonding and Thermoresponsive Surface-Mediated Reverse Transfection and Cell Detachment By Lei Tang, Wenguang Liu,* and Guipei Liu Gene delivery is one of the crucial steps for achieving successful gene therapy. With regard to delivery, a major concern is the ability to ferry the therapeutic gene to target cell population in a safe and effective mode. In liquid gene transfection (LGT) mode, DNA is condensed into nanometer-sized particles, which are then added into the culture media and the host cells take up the precipitated gene-laden nanocomposites. However the LGT cannot enable efficient localized gene delivery into targeted cells required for tissue engineering. [1,2] Recently, a new strategy termed reverse transfection or substrate-mediated delivery, where DNA was immobilized onto a surface of substrate, was reportedly adopted to improve the transfection efficiency due to direct and sufficient contact of substrate-supported DNA or siRNA with adherent cells. [3,4] In this context, the efficient and localized gene transfer is closely related to the cell-culture substrate. [5] As soft and wet materials, hydrogels have been widely used as extracellular substrate mimics for cell culture in tissue engineering. However, the actual utilization has been seriously impeded by their poor mechanical properties. To solve this issue, several groups developed double-network (DN) hydrogel, [6] exfoliated inorganic clay crosslinked nanocomposite hydrogel, [7] and macromolecular microsphere composite hydrogel, [8] which demonstrated very high strength. Nevertheless, these hydrogels cannot be used as the matrices for reverse transfection because of lacking the ability to anchor DNA on the surface. So far, very few studies have been conducted on exploring hydrogels as substrates for gene delivery, and the reported gene-tethered hydrogels were only coated onto the surface of metal stent or the mechanical strength was not noticed. [4,9] Herein, we presented a facile paradigm, whereby the mechanically strong hydrogels with integrated functional surface of hydrogen bonding and thermoresponsiveness were fabricated to meet the requirements of both DNA binding and gene- modified cell release. The novelties of these H-bonded and temperature sensitive (HT) hydrogels lie in that the H-bonding motifs located in the bulk and at surface respectively could confer strengthening and DNA anchoring abilities to the matrices without involving complicated immobilization procedure, [9] and temperature sensitiveness could be used to tune the attachment and detachment of gene modified cells, which could serve as seed cells for tissue engineering. Due to its ability to withstand high loading and release gene in situ, this HT hydrogel tethering gene on surface is expected to be directly implanted in vivo for gene therapy of load-bearing degenerative soft tissues such as disc degeneration disease, [10] which afflicts millions of people across the world. Additionally, the high strength offers another merit in withstanding mechanical handling for in vitro cell culture. The HT hydrogels were fabricated by crosslinking copolymer- ization of N-isopropylacrylamide (NIPAAm), 2-vinyl-4,6-diamino- 1,3,5-triazine (VDT), and the crosslinker poly(ethylene glycol) diacrylate (PEGDA, Mn ¼ 575). PNIPAAm components in hydrogels allowed for the attachment and detachment of cultured cells by temperature change without the need for enzymatic treatment. [11,12] It was established that VDT moieties were capable of forming complementary hydrogen bonding with base pairs of nucleic acid. [13–15] Thus, it is anticipated that VDT moieties on the surface of HT hydrogels could be hydrogen-bonded with DNA to realize local gene delivery. Owing to rather low solubility of VDT monomer in water, photopolymerization of HT hydrogels was conducted in DMSO. PEGDA acting as a crosslinker could improve not only the hydrophilicity, but also the cytocompatibility of hydrogels. The HT hydrogels were prepared by varying NIPAAm/VDT ratios. Table 1 demonstrates that the mechanical properties of hydrogels vary considerably with respect to the composition ratios. cr-PNIPAAm and HT gels with higher NIPAAm/VDT ratios (PNV2-1, PNV3-2, and PNV1-1) exhibit very weak proper- ties, whereas increasing VDT content tremendously enhances the mechanical properties. For PNV1-2, knotting, bending, and elongation cannot damage it (Fig. 1). The gel becomes tough and ductile with introducing an appropriate ratio of VDT. Under the same crosslinker dosage, the PNV1-3 hydrogel shows several orders of magnitude higher tensile strength (1.29 MPa) and modulus (750kPa; Table 1) than other hydrogels. This result indicates that incorporation of VDT can remarkably improve the mechanical strength of the hydrogels. The enhanced mechanical properties are ascribed to the H-bonding strengthening effect of VDT, which was shown to form relatively rigid six-membered ring structure by stacking of an extended aromatic core. [16] It was reported that PVDT provided apolar microenvironment consist- ing of less regularly arranged diaminotriazine (DAT) residues at the binding sites for hydrogen bonding formation in water. [17] Therefore, the efficient formation of hydrogen bonding between DAT in water eventually enhances the mechanical strength of hydrogels, as schematically depicted in Figure 1. It is noted that the tensile strengths of PNV1-5 and cr-PVDT are inferior to that of COMMUNICATION www.advmat.de www.MaterialsViews.com [*] Prof. W. G. Liu, L. Tang, G. P. Liu School of Materials Science and Engineering Tianjin Key Laboratory of Composite and Functional Materials Tianjin University, Tianjin 300072 (P. R. China) E-mail: [email protected] DOI: 10.1002/adma.200904016 2652 ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Mater. 2010, 22, 2652–2656

High-Strength Hydrogels with Integrated Functions of H-bonding and Thermoresponsive Surface-Mediated Reverse Transfection and Cell Detachment

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High-Strength Hydrogels with Integrated Functions ofH-bonding and Thermoresponsive Surface-MediatedReverse Transfection and Cell Detachment

By Lei Tang, Wenguang Liu,* and Guipei Liu

[*] Prof. W. G. Liu, L. Tang, G. P. LiuSchool of Materials Science and EngineeringTianjin Key Laboratory of Composite and Functional MaterialsTianjin University, Tianjin 300072 (P. R. China)E-mail: [email protected]

DOI: 10.1002/adma.200904016

� 2010 WILEY-VCH Verlag Gmb

Gene delivery is one of the crucial steps for achieving successfulgene therapy. With regard to delivery, a major concern is theability to ferry the therapeutic gene to target cell population in asafe and effective mode. In liquid gene transfection (LGT) mode,DNA is condensed into nanometer-sized particles, which are thenadded into the culture media and the host cells take up theprecipitated gene-laden nanocomposites. However the LGTcannot enable efficient localized gene delivery into targeted cellsrequired for tissue engineering.[1,2] Recently, a new strategytermed reverse transfection or substrate-mediated delivery, whereDNAwas immobilized onto a surface of substrate, was reportedlyadopted to improve the transfection efficiency due to direct andsufficient contact of substrate-supported DNA or siRNA withadherent cells.[3,4] In this context, the efficient and localized genetransfer is closely related to the cell-culture substrate.[5] As softand wet materials, hydrogels have been widely used asextracellular substrate mimics for cell culture in tissueengineering. However, the actual utilization has been seriouslyimpeded by their poor mechanical properties. To solve this issue,several groups developed double-network (DN) hydrogel,[6]

exfoliated inorganic clay crosslinked nanocomposite hydrogel,[7]

and macromolecular microsphere composite hydrogel,[8] whichdemonstrated very high strength. Nevertheless, these hydrogelscannot be used as the matrices for reverse transfection because oflacking the ability to anchor DNA on the surface. So far, very fewstudies have been conducted on exploring hydrogels as substratesfor gene delivery, and the reported gene-tethered hydrogels wereonly coated onto the surface of metal stent or the mechanicalstrength was not noticed.[4,9]

Herein, we presented a facile paradigm, whereby themechanically strong hydrogels with integrated functional surfaceof hydrogen bonding and thermoresponsiveness were fabricatedto meet the requirements of both DNA binding and gene-modified cell release. The novelties of these H-bonded andtemperature sensitive (HT) hydrogels lie in that the H-bondingmotifs located in the bulk and at surface respectively could conferstrengthening and DNA anchoring abilities to the matriceswithout involving complicated immobilization procedure,[9] andtemperature sensitiveness could be used to tune the attachment

and detachment of gene modified cells, which could serve as seedcells for tissue engineering. Due to its ability to withstand highloading and release gene in situ, this HT hydrogel tethering geneon surface is expected to be directly implanted in vivo for genetherapy of load-bearing degenerative soft tissues such as discdegeneration disease,[10] which afflicts millions of people acrossthe world. Additionally, the high strength offers another merit inwithstanding mechanical handling for in vitro cell culture.

The HT hydrogels were fabricated by crosslinking copolymer-ization ofN-isopropylacrylamide (NIPAAm), 2-vinyl-4,6-diamino-1,3,5-triazine (VDT), and the crosslinker poly(ethylene glycol)diacrylate (PEGDA, Mn¼ 575). PNIPAAm components inhydrogels allowed for the attachment and detachment of culturedcells by temperature change without the need for enzymatictreatment.[11,12] It was established that VDTmoieties were capableof forming complementary hydrogen bonding with base pairs ofnucleic acid.[13–15] Thus, it is anticipated that VDTmoieties on thesurface of HT hydrogels could be hydrogen-bonded with DNA torealize local gene delivery.

Owing to rather low solubility of VDT monomer in water,photopolymerization of HT hydrogels was conducted in DMSO.PEGDA acting as a crosslinker could improve not only thehydrophilicity, but also the cytocompatibility of hydrogels. TheHT hydrogels were prepared by varying NIPAAm/VDT ratios.

Table 1 demonstrates that the mechanical properties ofhydrogels vary considerably with respect to the compositionratios. cr-PNIPAAm and HT gels with higher NIPAAm/VDTratios (PNV2-1, PNV3-2, and PNV1-1) exhibit very weak proper-ties, whereas increasing VDTcontent tremendously enhances themechanical properties. For PNV1-2, knotting, bending, andelongation cannot damage it (Fig. 1). The gel becomes tough andductile with introducing an appropriate ratio of VDT. Under thesame crosslinker dosage, the PNV1-3 hydrogel shows severalorders of magnitude higher tensile strength (1.29MPa) andmodulus (750 kPa; Table 1) than other hydrogels. This resultindicates that incorporation of VDT can remarkably improve themechanical strength of the hydrogels. The enhanced mechanicalproperties are ascribed to the H-bonding strengthening effect ofVDT, which was shown to form relatively rigid six-membered ringstructure by stacking of an extended aromatic core.[16] It wasreported that PVDT provided apolar microenvironment consist-ing of less regularly arranged diaminotriazine (DAT) residues atthe binding sites for hydrogen bonding formation in water.[17]

Therefore, the efficient formation of hydrogen bonding betweenDAT in water eventually enhances the mechanical strength ofhydrogels, as schematically depicted in Figure 1. It is noted thatthe tensile strengths of PNV1-5 and cr-PVDTare inferior to that of

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Table 1. Contact angles and mechanical properties of hydrogels

Hydrogel Contact angle [deg] Tensile strength [kPa] Elongation at break [%] Young’s Modulus [kPa] Compression

20 8C 37 8C Stress [kPa] Fracture strain [%]

pH¼ 7.4

cr-PNIPAAm 42.4� 0.9 44.2� 1.7 7.9� 0.6 45.8� 4.2 17.4� 3.1 34.8� 3.6 44.6� 9.0

PNV2-1 51.0� 1.7 59.1� 1.1 7.7� 1.0 36.6� 7.8 21.3� 1.6 70.8� 16.3 57.2� 4.0

PNV3-2 56.2� 1.6 60.4� 1.0 7.4� 1.2 46.1� 12.0 16.3� 1.4 119.3� 29.9 57.7� 9.6

PNV1-1 62.7� 1.8 64.8� 1.2 9.1� 0.5 41.5� 5.2 22.1� 1.9 536.6� 65.1 73.7� 2.3

PNV1-2 71.3� 0.8 75.9� 0.9 470.3� 34.7 317.5� 46.6 150.5� 26.7 3169.7� 110.1 100.1� 8.1

PNV1-3 77.5� 1.2 80.7� 0.8 1290.3� 123.9 176.1� 36.9 746.8� 100.4 2816.1� 756.4 89.6� 22.7

PNV1-5 86.3� 1.0 88.2� 1.0 872.7� 23.0 170.0� 5.5 513.7� 24.9 3996.4� 1365.7 91.1� 15.8

cr-PVDT [a] [a] 504.0� 3.8 158.9� 16.0 319.2� 30.8 3725.0� 111.4 88.0� 1.2

[a] An accurate contact angle for cr-PVDT was not available due to phase separation-induced large surface roughness of this gel.

PNV1-3. An explanation is that the network of hydrogels becomesheterogeneous due to the occurrence of phase separation athigher contents of hydrophobic VDTmoieties, which somewhatleads to the increased brittleness. Therefore, the mechanicalproperties can also be adjusted by varying PNIPAAm/VDTratios.Another attractive feature is that the HT hydrogel alsodemonstrates a high compression fracture stress at an optimumNIPAAm/VDT ratio at pH 7.4. The compression stress of HThydrogel reaches to 3.2MPa, more than 100 times higher thanthat of cr-PNIPAAm (34.8 kPa), when NIPAAm/VDT is adjustedto 1/2, close to that of cr-PVDT (Table 1). Furthermore, thePNV1-2 can recover its original shape after the load is released.(Supporting Information, Fig. S1).

The mechanical properties were also found to strongly rely onthe pH of media (Supporting Information, Table S1). A severedecrease in the tensile strength andmodulus occurs when the pHvalue is decreased to 5.0 and 3.8. Note that in this case thecr-PNIPAAm, PNV2-1, PNV3-2, and PNV1-1 are too weak to bemeasured on the mechanical tester. These results offer furtherevidence that H-bonding of PVDT is responsible of the enhanced

Figure 1. Photographs demonstrating the robustness of PNV1-2 with an ewithstand knotting (A), bending (B), and elongation (C). D) Swelling behavior oat different pHs. E) schematic diagram depicting the pH-responsiveness of

Adv. Mater. 2010, 22, 2652–2656 � 2010 WILEY-VCH Verlag G

strength of gels. At a pH lower than 5.15, pKa of PVDT,[18] DATresidues are protonated, causing the breakage of hydrogenbonded network to a varied extent (Fig. 1E). Moreover, muchmore H-bonding association is damaged due to the increasedextent of protonation at a lower pH. Hence, more seriousdeterioration of the mechanical properties occurs at pH 3.8. Avisual inspection of gel finds that the gel becomes more swollenand transparent while pH drops due to the electrostatic repulsionbetween neighboring NH3

þ (Fig. 1D). It is noteworthy thatthe pH sensitiveness of HT gels is reversible.

The appearance of HT hydrogels with varied compositions canbe distinguished visually (Supporting Information, Fig. S2). Thegels remain highly transparent in the selected range of NIPAAm/VDT ratios at 20 8C and pH 7.4; however cr-PVDT gel becomestranslucent. The light transmission and equilibrium watercontent (EWC) of hydrogels were determined at differenttemperatures using a microbalance and a UV–vis spectrometer,respectively (Fig. S3 and S4). As expected, cr-PVDT hydrogel isinsensitive to temperature during heating, while HT hydrogelsexhibit thermoresponsive behavior due to thermally induced

xcellent ability tof PNV1-2 hydrogelPNV1-2 hydrogel.

mbH & Co. KGaA, Wein

hydration and dehydration of PNIPAAm. Aclose inspection finds that the light transmis-sions of cr-NIPAAm, PNV2-1, PNV3-2, andPNV1-1 gels show mild discontinuous transi-tions at 31, 30, 29, and 28 8C, respectively,evidencing introducing hydrophobic VDTshifts the phase transition toward lowertemperature. At higher contents of VDT, thereappears a continuous transition of lighttransmission. The vanishing of discontinuityis originated from the incorporation oftemperature-insensitive moieties and highercrosslinking contents used.[19] For EWC, allhydrogels shows continuous transition alongwith temperature change for the same reasonas above mentioned. A similar phenomenonwas also reported previously.[20] These resultsmanifest that turbidity is more sensitive totemperatures at high NIPAAm/VDT ratios.

Another general feature is that uponincreasing the proportion of VDT, EWC andlight transmission demonstrate a declining

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Figure 2. Phase-contrast microscopy images of PNV1-2 gel-inducedCOS-7 cell adhesion at 37 8C (A) and detachment at 20 8C (B). C) In vitrogene transfection efficiency of hydrogels and cell detachment efficiency.Data represent mean� standard deviation (SD; n¼ 3).

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trend, suggesting that raising VDT/NIPAAm ratios leads to theincreased hydrophobicity of hydrogels.

Table 1 shows the temperature dependence of the water contactangles on a hydrogel surface. Clearly, while the temperature israised from 20 to 37 8C (two temperatures for following celldetachment and attachment), the surface of hydrogels becomesmore hydrophobic. We also find that increasing the VDT ratiosleads to the enhancement in the hydrophobicity of the surface.The contact angle of PNV1-5 is as high as 868, lower than that ofpolystyrene, 948,[21] but larger than 808, the contact angle ofpoly(l-lactide), a hydrophobic polymer with poor cell affinity.[22]

Note that we cannot obtain an accurate contact angle of cr-PVDTdue to phase-separation-induced large surface roughness of thisgel. Nonetheless, based on the above analysis, it is rational toassume that cr-PVDT is more hydrophobic.

In cell-culture experiments we found that the viable cellsadhered on cr-PVDT hydrogel after 48 h of culture did not presenttypical spread morphology, being quite round and sparselydistributed, which suggests poor cell affinity. Most of the cellsdetached from the cr-PVDTwere possibly washed off by pipetting.The poor cell adhesion on cr-PVDT hydrogel is attributed to thehigh hydrophobicity of PVDT as well as higher cytotoxicity (Fig.S5). Comparatively, the cells cultured on the HT hydrogelsassume the typical spread morphology. It is obvious thatcopolymerization of NIPAAm can improve the cell affinity ofPVDT hydrogels. We detached the cells by short-time cooling ofculture medium. After incubation at 20 8C for 1 h, the cellsdetached from the hydrogels were harvested and cultured in anew 48-well plate. The cell-detachment efficiency of the hydrogelsis in the range from 70% to 80% (Fig. 2). The morphologies ofCOS-7 on the surface of PNV1-2 demonstrate that the cells can beefficiently released by cooling (Fig. 2A and 2B). It is consideredthat the dynamic process of surface hydration switch and swellingleads to the decrease of adhesion strength.[23,24] Additionally,there appears a reduction trend of detachment efficiency with theincrease in the content of VDT, as expected, whereas the higherdetachment efficiency of cr-PVDT does not arise from thetemperature change but from poorer cell affinity. We find thatafter the cells detached from the thermoresponsive hydrogelswere re-cultured into a new plate, they resumed to normalgrowth. This reveals that the viability of the detached cells can wellbe maintained.

In our previous work, we showed that PVDT-based polymerswere capable of condensing DNA by hydrogen bonding associa-tion with base pairs and thereby achieving an efficient transfec-tion.[25] However, the rather low solubility of PVDT limits itsapplication in gene delivery. Herein, we resorted to PVDT-basedhydrogels mediatied reverse gene transfection. Note that nakedDNA and PVDT/DNA complex particles were respectivelyanchored on the surface for gene-transfection evaluation in thisstudy. The size and zeta potential of PVDT/DNA complexparticles (PVDT/DNA weight ratio¼ 0.25) respectively measuredas 1.3mm and �4.2mV, was reported in our previous work.[25,26]

The adsorption efficiency of pDNA on the surface of hydrogels isenhanced with an increment of the content of VDT owing to theenrichment of more H-bonding motifs on the surface(Supporting Information, Fig. S6), as demonstrated by thehigher contents of N element in X-ray photoelectron spectroscopy(XPS) upon raising the VDTratio (Supporting Information, Table

� 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Mater. 2010, 22, 2652–2656

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Figure 3. Fluorescence images of cr-PNIPAAm adsorbed with YOYO-1 labeled pDNA (A),PNV1-1 adsorbed with YOYO-1 labeled pDNA (B), PNV2-1 adsorbed with YOYO-1 labeledcomplex particles (C), and PNV1-2 adsorbed with YOYO-1 labeled complex particles (D).

S2). In order to verify the adsorption of pDNA and complexparticles on the surface, the hydrogels coated withYOYO-1-labeled pDNA and complex particles were imaged byfluorescence microscopy (Fig. 3). With the same amount ofpDNA, the image of cr-PNIPAAm (A) shows very little fluorescence;while stronger fluorescence is observed on the surface of PNV1-1(B). Under the same dosage of PVDT/pDNA complex particles, itis noticeable that PNV1-2 presents a higher intensity offluorescence than other hydrogels due to its inclusion of morecontent of VDT (Fig. 3D).

From the reverse transfection results of hydrogels, as measuredby recording luciferase activity in COS-7 cells (Fig. 2C), one can seethat the transfection efficiencies go up with an increment of VDTcontent in the hydrogels because of the increased amount ofadsorbed DNA on the surface. For naked pDNA binding, PNV1-2achieves approximately 100-fold higher transfection efficiencythan cr-PNIPAAm-lacking VDT moieties. Furthermore, thetransfection levels of complex particles on the surface of thehydrogels are 1.7–7.6-fold of those of adsorbed naked DNA.Similar to the liquid phase transfection, condensing DNA by avector can aid in gene transport. The optimum efficiency achievedby the complex particle-adsorbed surface of PNV1-2 hydrogel is2.2� 106 RLUmg�1, higher than the liquid phase transfection ofPVDT-based polymers.[25] However, the cr-PVDT showed arelatively low transfection efficiency due to its high cytotoxicity.Collectively, PNV1-2 possesses the excellent mechanical proper-ties, better cell adhesion, detachment ability, and highertransfection efficiency. Encouragingly, HT hydrogels presentlow cytotoxicity to COS-7 cells (Supporting Information, Fig. S5).

In conclusion, we have successfully prepared hydrogen-bonded and thermoresponsive hydrogels with both high tensileand compressive strengths, which are rarely found in the reportedstrong hydrogels.[6–8,27] The hydrogen bonding in the bulk of HT

Adv. Mater. 2010, 22, 2652–2656 � 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Wein

hydrogels acted to strengthen the mechanicalproperties of the gels, and the hydrogenbonding motifs and temperature sensitivenesson the surface of hydrogel substrate offer aplatform for reverse gene transfection and cellrelease. This mechanically strong temperature-sensitive hydrogel anchoring gene on thesurface holds a great potential as a integratedfunctional scaffold for the harvest of gene-modified seed cells for tissue engineeringwithout unfavorable enzyme treatment as wellas the local gene delivery for the regenerationand repair of load-bearing soft tissues. Themethod reported here can become be a facilestrategy to construct multifunctional high-strength soft-wet tissue engineering scaffoldsby copolymerizing monomer containing hydro-gen bonding motifs with other hydrophilicmonomer.

Experimental

Preparation of Hydrogels: An appropriate amountof NIPAAm, VDT, PEGDA, and IRGACURE 2959photoinitiator (2% w/w, relative to the total monomermass), were dissolved in DMSO. The mixture was

cast into disc molds (diameter 12.6mm, thickness 1.6mm) or tubularmolds (inner diameter 10mm, length 12mm), and the photopolymeriza-tion was carried out for 20min in a crosslink oven (XL-1000 UV Crosslinker,Spectronics Corporation, NY, USA). The hydrogels obtained were washedthoroughly with distilled water to remove the unreacted monomer andsolvent. Herein, crosslinked PNIPAAm hydrogel (cr-PNIPAAm), copolymerhydrogels (PNVx–y) and crosslinked PVDT hydrogel (cr-PVDT) weresynthesized (x-y represents the NIPAAm/VDT weight ratio; Table S3). Thecontent of PEGDA was fixed for all the formulations. FTIR-ATR spectro-scopy and the XPS spectra confirmed the formation of P(NIPAAm-co-VDT)HT hydrogels (Supporting Information, Fig. S7, Table S2).

Measurement of Mechanical Properties: The mechanical properties ofhydrogels were measured on an electromechanical tester (LLY206B,Laizhou electric instrument Co. ltd., China) at room temperature.Hydrogels were fully equilibrated in 0.1M PBS buffer solutions(pH¼ 3.8, 5.0, 7.4) and cut into rectangular pieces with20mm� 2mm� 0.50mm dimensions. Crosshead speed was set at10mmmin�1, and three specimens were tested for each hydrogel sample.For compression tests, the samples were cut into cylinders (10mm indiameter and 8mm thick) and measured on EnduraTEC ELF 3200 materialtesting.

Detachment of Cells Cultured on Hydrogels: For cell detachment, COS-7cells were cultured by a protocol (presented in the SupportingInformation), and seeded into a 48-well plate coated with hydrogels at adensity of 4� 104 cells per well and cultured at 37 8C for 48 h under ahumidified atmosphere of 5% CO2. Then, the cell-attached gels were takenout and placed into a new plate for cooling treatment at 20 8C for up to 1 hto make the NIPAAm-based hydrogels rehydrated. After that, the culturemedium containing the detached cells and the rehydrated hydrogelsattached with the residual cells were transferred to new wells, respectively.The cell detachment was evaluated by MTT assay and the detachmentefficiency (%) was defined as the absorbance (at 570 nm) of detached cells(harvested from culture medium) divided by that of total cells on thesurface of hydrogel (detached cells plus cells left on the rehydratedhydrogels).

Adsorption of Plasmid DNA and PVDT/pDNA Complex Particles for GeneDelivery: Sterilized hydrogel discs were transferred to a 48-well plate intowhich 400mL DMEM was added for further treatment. The solutions of

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PVDT/pDNA complex particles, formed based on the protocol in ourprevious study [25], and naked pDNA pGL3-control (2mg pDNA diluted in100mL DMEM) were separately added to each hydrogel-coated well. After23 h incubation at 20 8C and then 1 h at 37 8C (hydrophobic state hydrogel),each hydrogel disc was washed gently with 500mL DMEM once. Then,COS-7 cells were seeded on the rinsed hydrogels for reverse transfection.24 h later, the medium was replaced with fresh DMEM plus 10% FBS,followed by further incubation for 24 h to express the luciferase reportergene. The cells were released from the gels by cooling at 20 8C for 1 h.Immediately, the released cells were centrifuged for 5min to remove themedium and washed with PBS twice. The cells were treated for 15min with150mL of reporter lysis buffer (RLB, Promega) followed by freeze-thawcycles to ensure complete lysis. Luciferase activity in the transfected cellswas measured according to the reported method [25].

Acknowledgements

This work was financially supported by the National Natural ScienceFoundation of China (grant 30770587) and the Program for New CenturyExcellent Talents in University. We also thank Dr. Mike Cao at the Universityof Washington for useful discussions. Supporting Information is availableonline from Wiley InterScience or from the author.

Received: November 24, 2009

Revised: January 8, 2010

Published online: May 20, 2010

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