Immobilization of Stimuli-Responsive Nanogels onto Honeycomb

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Immobilization of Stimuli-Responsive Nanogels onto Honeycomb Porous Surfaces and Controlled Release of Proteins A. S. De León,† M. Molina,‡ S. Wedepohl,‡ A. Muñoz-Bonilla,§ J. Rodríguez-Hernández,*,† and M. Calderón*,‡ †

Instituto de Ciencia y Tecnología de Polímeros (ICTP), Consejo Superior de Investigaciones Científicas (CSIC), C/Juan de la Cierva 3, 28006 Madrid, Spain ‡ Institute of Chemistry and Biochemistry, Freie Universität Berlin, Takustr. 3, 14195 Berlin, Germany § Departamento de Química Física Aplicada, Facultad de Ciencias, Universidad Autónoma de Madrid, C/Francisco Tomás y Valiente 7, Cantoblanco, 28049 Madrid, Spain S Supporting Information *

ABSTRACT: In this article, we describe the formation of functional honeycomblike porous surfaces fabricated by the breath figures technique using blends of either amino-terminated poly(styrene) or a poly(styrene)-b-poly(acrylic acid) block copolymer with homopoly(styrene). Thus, the porous interfaces exhibited either amino or acid groups selectively located inside of the holes, which were subsequently employed to anchor stimuli-responsive nanogels by electrostatic interactions. These nanogels were prepared from poly(N-isopropylacrylamide) (PNIPAM) cross-linked with dendritic polyglycerol (dPG) and semi-interpenetrated with either 2-(dimethylamino)ethyl methacrylate (DMAEMA) or 2acrylamido-2-methyl-1-propanesulfonic acid (AMPS) to produce positively and negatively charged nanogel surfaces, respectively. The immobilization of these semi-interpenetrated networks onto the surfaces allowed us to have unique stimuliresponsive surfaces with both controlled topography and composition. More interestingly, the surfaces exhibited stimuli-responsive behavior by variations on the pH or temperature. Finally, the surfaces were evaluated regarding their capacity to induce a thermally triggered protein release at temperatures above the cloud point temperature (Tcp) of the nanogels.

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INTRODUCTION Surface modification of materials to obtain specific properties different than in the bulk is receiving more and more attention in the last years.1−4 Particularly, in the biomaterials field, there is a great interest to design biocompatible surfaces for biomedical applications.5−7 The control and modification of the surface without changing the bulk properties is critical for the design of these novel biomaterials. In particular, molecularrecognition systems require a 3D structure whose chemical functionalities and structure can be precisely controlled. However, it is difficult to find generalities in this subject due to the large variety of organisms studied and incubation conditions. In addition, in the polymer field, it still remains a challenge to develop simple and economical methods to physically or chemically modify the plastics surface.8 In this sense, the breath figures approach presents a good alternative to yield porous surfaces with controlled topography and composition. These platforms have been widely used for different bioapplications, such as cell growth processes or protein immobilization.9−12 In the last years, special interest has been devoted to the fabrication of nanogels. These high molecular weight crosslinked polymers combine the characteristics of nanoparticles © XXXX American Chemical Society

with those of cross-linked macroscopic gels, to yield soluble or dispersible particles within the useful size range between 20 and 200 nm.13 Stimuli-sensitive functional groups can be introduced within the nanogel internal structure, to render molecules that can shrink or swell rapidly by expelling or absorbing water in response to external stimuli such as temperature, pH, or magnetic fields.14−17 Therefore, the use of stimuli-responsive nanogels as active nanocarriers offers an interesting alternative as smart delivery systems in the optimization of biomedical therapies.18 Among different stimuli-sensitive delivery modalities, temperature-sensitive drug delivery systems offer great potential due to their versatility and tunability of the transition temperatures. In particular, poly(N-isopropylacrylamide) (PNIPAM) nanogels present a phase transition temperature (Tcp) near to human body temperature.18 Within this context, the aim of this publication concerns the preparation of polymeric surfaces with controlled topography (obtained by the breath figures approach) and functionality provided by the immobilization of nanogels onto the pores. Received: November 25, 2015 Revised: January 19, 2016

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DOI: 10.1021/acs.langmuir.5b04166 Langmuir XXXX, XXX, XXX−XXX

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Poly(styrene)-b-poly(acrylic acid) (PS19-b-PAA10) was synthesized via ATRP according to previously reported procedures. Briefly, first a linear PS was synthesized by ATRP, and subsequently used as macroinitiator to polymerize tert-butyl acrylate. Then, acrylic acid groups were obtained by adding trifluoroacetic acid (excess) in dichloromethane thus deprotecting the tert-butyl groups. Poly(styrene) amino terminated (PS45−NH2) was synthesized from a PS macroinitiator also made by ATRP (PS45−Br) by making it react with ethylenediamine (excess) in DMF overnight. Afterward, in a typical procedure, porous films were prepared by drop casting a mixture of PS and the additive, having either carboxylic acid groups, that is, PS19-bPAA10, or PS45−NH2, in a chloroform solution under high relative humidity conditions (80−99%) and room temperature inside a closed chamber. More details about the precise fabrication of these surfaces have been described elsewhere.22,23 Immobilization of Nanogels. Surfaces were immersed in a solution of HEPES 10 mM (pH 6) containing 0.5 mg mL−1 of the different SIPN or the PNIPAM nanogel at 25 or 45 °C. Different incubation times, ranging from 5 min to 2 h, were tested. Then, the solution was recovered and the surfaces were thoroughly washed in HEPES 10 mM (pH 6) buffer and dried for fluorescence microscopy analysis. To study the pH responsiveness of the SIPN, complementary assays were done by performing incubations at pH 10 and/or subsequent immersion and washing steps in buffers at pH 6 or 10. Encapsulation and Release of BSA-Rh. Bare and SIPN PNIPAM nanogels were loaded with BSA-Rh via swelling-diffusion process. Briefly, 10 mg mL−1 nanogels were swollen in a protein solution (10 mg mL−1) for 24 h at 4 °C. The solutions were purified using Vivaspins 300000 (10 min at 6000 rpm; Sartorius AG, Göttingen, Germany). The concentration of the free protein was determined by UV measurements, and the protein loading capacity of the nanogels was calculated. After purification, BSA-Rh loaded nanogels were incubated to be immobilized for 2 h onto the surfaces as described in the step before. Then, the surfaces were dried and immediately immersed in 2 mL of HEPES 10 mM (pH 6) buffer solution for 1 h at 25 or 45 °C. Then, surfaces were washed and dried for fluorescence microscopy analysis. Measurements. 1H NMR analyses were performed using a Bruker 400 MHz NMR spectrometer. The sample preparation, in which 8 mg of nanogel had been dissolved in 0.8 mL of D2O, was prepared 24 h prior to the measurement. Fourier transform infrared (FTIR) spectroscopy analysis was carried out using a Bruker IFS 66 FT-IR spectrophotometer in the range of 4000−500 cm−1. The nanogel particle sizes and polydispersity were measured at temperatures ranging from 25 to 50 °C by dynamic light scattering (DLS) using a Nano-ZS 90 Malvern instrument equipped with a He−Ne laser (λ = 633 nm) under scattering of 173°. All the samples were maintained for stabilization at the designed temperature for 5 min before testing. The samples were prepared by dissolving 1 mg of dry nanogel in 1 mL of HEPES buffer solution pH 6 one day prior to the experiments. Particle size and size distribution are given as the average of three measurements from the volume distribution curves. Cloud point temperature (Tcp) was measured on a Cary 100 Bio UV−vis spectrophotometer equipped with a temperature-controlled, sixposition sample holder. HEPES buffered at pH 6 nanogels solutions (1 mg mL−1) were heated at 0.2 °C min−1 while monitoring both the transmittance at 500 nm (1 cm path length) and the solution temperature (from 25 to 65 °C), as determined by the internal temperature probe. The Tcp of each nanogel was determined using the minimum of the first derivate of transmittance vs temperature. Scanning electron microscopy (SEM) micrographs were taken using a Philips XL30 instrument with an acceleration voltage of 25 kV. The samples were coated with gold−palladium (80/20) prior to scanning. Atomic force microscopy (AFM) measurements were conducted on a Multimode NanoscopeIVa, Digital Instrument/Veeco apparatus operated in tapping mode under ambient conditions. Fluorescence microscopy (FM) assays were performed using a Carl Zeiss AxioVert, compact light source HXP 120C system (Carl Zeiss MicroImaging GmbH, Jena, Germany). Images were taken at different magnifications using ×40 and ×63 objectives and the corresponding set of filters for

Nanogels immobilized at interfaces are particularly interesting due to their high cargo capacities, their varieties and high density of functional groups, and their capacity to respond to different external stimuli.19 These nanogels will serve not only as functional supports but also as macromolecular carriers of active molecules (such as imaging probes, proteins, drugs, or even genes) allowing a specific controlled release only under certain ambient conditions.

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EXPERIMENTAL SECTION

Materials. The following chemicals were used as purchased: acryloyl chloride (96% Fluka), triethylamine (TEA, 99%, Acros), dimethyl sulfoxide (DMSO, Scharlau), chloroform (Scharlau), dry dimethylformamide (DMF, 99.8%, Acros), N-isopropylacrylamide (NIPAm, 97%, Aldrich), ammonium persulfate (APS, 98%, Aldrich), sodium dodecyl sulfate (SDS, 98%, Acros), tetramethylenethylendiamine (TEMED, 99%, Aldrich), 2-[4-(2-hydroxyethyl)-1-piperazine]ethanesulfonic acid (HEPES, 99%, Acros), 2-(dimethylamino)ethyl methacrylate (DMAEMA, 99%, Acros), 2-acrylamido-2-methyl-1propanesulfonic acid (AMPS, 99%, Aldrich), fluorescein isothiocyanate (FITC, 98%, Aldrich), sodium phosphate dibasic and monobasic (Aldrich), poly(styrene) (average Mw = 350 000, Aldrich), and Sephadex G 25 Fine (GE Healthcare). Dendritic polyglycerol (dPG) with average Mw of 10 kDa (PDI = 1.3) was synthesized according to previously reported methodologies. The amine functionalization of dPG was performed as previously reported.20 Benzoylated regenerated cellulose membrane (2 kDa MWCO, Sigma-Aldrich), regenerated cellulose membrane (50 kDa MWCO, SpctraPor), and bovine serum albumin rhodamine B labeled (BSA-Rh, Life Technologies) were used as received. Synthesis of Fluorescent Semi-Interpenetrated Networks. Dendritic Polyglycerol Fluorescein Isothiocyanate-Labeled (dPGFITC). FITC (4.7 mg) was dissolved in a small amount of DMSO (i.e., 100 μL). Then, 60 mg of dPG-NH2 (∼13.5 amino groups (10%)) and 2 mL of phosphate buffered saline (PBS, 10 mM, pH 7.4) were added. Reaction took place at room temperature in 1 h. Successful coupling of FITC was checked by thin layer chromatography (TLC). Product was purified by size exclusion chromatography (SEC) with Sefadex G25 Fine and then lyophilized. dPG-FITC 10% Acrylated (dPG-FITC-Ac). Dried dPG-FITC (30.0 mg) was added in a flask under Ar atmosphere with dry DMF (5 mL) at 80 °C under stirring. Then, temperature was set to 0 °C and TEA was added (11.3 μL). Acryloyl chloride (3.9 μL) was added dropwise, and the reaction was carried out under stirring for 4 h. Product was purified by dialysis membrane (MWCO 2 kDa) against water for 72 h. PNIPAM Nanogels Cross-Linked with dPG-FITC-Ac (33 wt %). PNIPAM-dPG nanogels were synthesized according to previously reported methods.21 Briefly, 55.4 mg of NIPAM, 27.3 mg of dPGFITC-Ac, SDS (1.8 mg), and APS (2.8 mg) were dissolved in 5 mL of distilled water. Argon was bubbled into the reaction mixture for 15 min. The reaction mixture was transferred into a hot bath at 70 °C, and polymerization was activated after 5 min with the addition of catalytic amount of TEMED (120 μL). The mixture was stirred at 500 rpm for at least 4 h. The products were purified by dialysis membrane (MWCO 50 kDa) against water for 48 h and then lyophilized to yield the nanogels as a yellowish solid. Semi-Interpenetrated Networks (SIPN) of PNIPAM Nanogels with Either DMAEMA or AMPS. APS (1.0 mg) and DMAEMA (4.0 μL) or AMPS (4.9 mg) were mixed in 1 mL of water under Ar atmosphere. When a homogeneous mixture was obtained, dry PNIPAM nanogel (20.0 mg) was added. The mixture was left for 4 h to allow the diffusion of the monomer inside of the nanogel, so it can be semiinterpenetrated. Stirring was not necessary. Then, 10 μL of TEMED was added. Reaction took place at 0 °C overnight. The product was then purified by SEC and then lyophilized to yield fluorescent labeled SIPN networks. More details about synthesis and purification have been described elsewhere.21 Preparation of the Additives and Fabrication of Honeycomb-Patterned Films by the Breath Figures Technique. B


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Langmuir Scheme 1. Synthesis of Semi-Interpenetrated Nanogelsa

a

(a) Coupling with FITC of dPG-NH2 and further modification to obtain a terminal acrylate functional group; (b) PNIPAM nanogels formed using dPG-Ac as cross-linker (1); (c) semi-interpenetrated with AMPS (SIPN−) (2) or DMAEMA (SIPN+) (3). imaging: bright field, 38 HE eGFP or 4 HE Cy3. Data processing of the images were recorded with ImageJ and AxioVision SE64 Rel. 4.9.1 software.

unreacted (the amount of FITC was found to be around 19 nmol FITC per mg of bared nanogels). To form the nanogel, the dPG additionally required the surface functionalization with reactive acrylates. For that purpose, the amino groups present in the dPG were allowed to react with acryloyl chloride. A detailed description of the experimental conditions employed for the acrylation of dPG, formation of nanogels with NIPAM, and semi-interpenetration of the nanogels with DMAEMA or AMPS were made as described elsewhere.21 Nevertheless, it is worth to mention that the semi-interpenetration polymerization was left to proceed overnight to be sure that long enough polyelectrolyte chains are obtained in the nanogel which will be in turn required to establish electrostatic interaction with the porous surfaces. As a result, three different nanogels were synthesized prior to surface modification studies: PNIPAM cross-linked with dPG

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RESULTS AND DISCUSSION Preparation and Characterization of the Semi-Interpenetrated Nanogels with Thermal- and pH-Response. Thermo- and thermo/pH-responsive nanogels were prepared following the strategy illustrated in Scheme 1. Fluorescein isothiocyanate (FITC) was coupled to partially aminated dPG (dPG-NH2 10%, ∼13.5 NH2-groups) by means of the isothiocyanate functional groups. FITC allowed the detection of the nanogels localization at the surface upon immobilization by fluorescence microscopy. In this study, we introduced an average of two molecules of FITC per dPG, thus leaving a large amount of both amino and hydroxyl functional groups C


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studies with the nanogels in its collapsed state were carried out at 45 °C. Additionally, size and surface charge of the nanogels were determined above and below the Tcp. Table 1 summarizes the obtained results for PNIPAM-dPG, SIPN+, and SIPN−. All the measurements were done in HEPES buffer at pH 6.0 to be sure that the SIPN+ nanogel was protonated.25 Equally, at these pH values, the sulfonic acid groups comprised in the SIPN− nanogel were expected to be always deprotonated. The nanogels exhibited sizes between 160 and 220 nm. It is important to note that the sizes of both SIPN (∼175 and ∼219 nm) are higher than the size of the bare nanogel (∼158 nm). Most probably the chains of the polyelectrolyte semi-interpenetrated within the nanogel swell their structure thus leading to higher diameters. In agreement with the Tcp results described previously, the size of the nanogels decreased in all cases when temperature is increased at 45 °C (above the Tcp). This indicates a collapse of the nanogel structure upon water expulsion and the formation of molecular interactions between the PNIPAM chains. In addition to the nanogel size, zeta potential measurements confirmed that the semi-interpenetration reaction has been successfully achieved. As expected, the surface charge is modulated by the type of polyelectrolyte constructed. Therefore, SIPN+ provides surfaces with positive zeta potential values of 9.2 mV and SIPN− leads to negative surface charges (ζ = −12.2 mV). More interestingly, even when the SIPN exhibits surface charge above and below Tcp, the absolute surface charge value at 45 °C is higher than those measured at room temperature. In effect, due to the collapse of the PNIPAM molecules, the polyelectrolyte chains that remain soluble can be easily exposed to the solvent and accordingly increasing the surface charge density. Interestingly, the surface charge of the PNIPAM-dPG nanogels remained the same at both temperatures. The particle size distribution profiles for the SIPNs is shown in the Supporting Information. Immobilization of the Nanogels at the Pore Surface. The FITC-labeled semi-interpenetrated nanogels were immobilized onto the porous surfaces having complementary surface charge. More precisely, we prepared honeycomb porous films containing amino groups (NS) to immobilize SIPN− and

(PNIPAM-dPG, noncharge), PNIPAM-dPG semi-interpenetrated with DMAEMA (SIPN+), and PNIPAM-dPG semiinterpenetrated with AMPS (SIPN−). The PNIPAM-dPG composition was determined to be 66 wt % PNIPAM and 33 wt % dPG by 1H NMR.21 The effective semi-interpenetration was proven by FT-IR (Figure 1). In the case of SIPN−, the band of SO stretching can be seen at 1050 cm−1. For the SIPN+, an increment of the amide band is observed at 1721 cm−1.

Figure 1. FT-IR spectra of NG, SIPN−, and SIPN+.

Since the nanogels are based in PNIPAM, they are expected to exhibit a transition temperature collapsing above Tcp as depicted in Scheme 2. This has been investigated by measuring the UV-transmittance of the nanogels by varying the solution temperature. Within the resulting curves, the cloud point was calculated as the minimum value from the derivative of the transmittance. As depicted in Table 1, average Tcp values obtained were 38.1, 35.6, and 38.0 °C for PNIPAM-dPG, SIPN +, and SIPN−, respectively. In comparison with usual Tcp values observed for PNIPAM nanogels (around 33 °C), we observed a slight increase in the Tcp of the nanogels and SIPN functionalized with FITC. Most probably, these fluorescent labeled dPG nanogels exhibit a more hydrophilic character caused by the presence of FITC.24 Therefore, the rest of the

Scheme 2. Stimuli-Responsive Behavior of the Nanogels Prepareda

a PNIPAM-dPG (1) and SIPN− (2) exhibit thermoresponsive behavior so that upon heating the nanogel shrinks. SIPN+ (3) exhibits simultaneously pH and thermal response so that the surface charge and the swelling can be controlled varying the external pH and temperature.

D


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Langmuir Table 1. Cloud Point (Tcp), Size and ζ Potential of the PNIPAM-dPG and Both SIPNs below and above Tcp 25 °C

45 °C

nanogel

Tcp (°C)

size (nm)

PDI

ζ (mV)

size (nm)

PDI

ζ (mV)

PNIPAM-dPG SIPN+ SIPN−

38.1 35.6 38.0

158.7 175.6 219.5

0.272 0.300 0.327

−2.1 9.2 −12.2

116.1 150.5 196.2

0.145 0.247 0.154

−2.1 15.9 −18.2

Figure 2. Left: schematic illustration of the SIPN immobilization on either carboxylic acid (i) or amine (ii) functionalized porous surfaces. Right: (a) SEM and (b) FM micrographs of a NS with SIPN+; (c) SEM and (d) FM micrographs of AS with SIPN−. Scale bar: 10 μm.

porous films having carboxylic acid groups (AS)22,23 to immobilize the SIPN+ nanogels. PNIPAM-dPG was used as an uncharged control in both surfaces. The preparation of honeycomb porous films has been carried out by the breath figures approach consisting in the condensation of water vapor onto the surface of an evaporating polymer solution. During the evaporation of a solvent the solution/air interface is cooled and drives the condensation of water vapor. The condensed water vapor leads to the formation of water droplets so that upon complete evaporation of the polymer solution and the condensed droplets the surface exhibits micrometer scale pores. Our group has been involved in the preparation of such structures by using polymer blends of a homopolymer and a functional polymer (statistical copolymers or block copolymers) to prepare surfaces with particular surface chemistry. In particular, we demonstrated that hydrophilic functional groups tend to migrate toward the holes during the evaporation. Thus, these surfaces will specifically contain the functional groups located selectively inside of the holes.26,27 By using this strategy we prepared porous films exhibiting a hexagonal arrangement from two different blends. On the one hand, we prepared blends of poly(styrene) and amino-terminated poly(styrene) (PS45−NH2) to provide amino groups inside of the pores (NS). On the other hand, blends of poly(styrene) and an amphiphilic block copolymer (PS19-bPAA10) will serve to fabricate surfaces in which the pores are functionalized with carboxylic acid functional groups (AS). The SEM images of the surfaces containing either amino or carboxylic functional groups are depicted in Figure 2. In both cases, ordered micropores with sizes around 2−3 μm were obtained (see Experimental Section for further details).

We employed these surfaces to immobilize the positively and negatively charged SIPN using PNIPAM-dPG as control. For this purpose, the selected nanogel was dissolved in HEPES buffer 10 mM with a concentration of 0.5 mg mL−1 adjusting the pH to 6. The immobilization occurs by incubation for 1 h of either SIPN+ solution onto AS films or SIPN− solution onto NS surfaces. In addition, as a control experiment, a solution containing PNIPAM-dPG without any semi-interpenetrated polymer chains was also incubated in both surfaces. After the incubation, all surfaces were extensively washed with HEPES buffer. The success of the immobilization of the SIPN was followed by FM. Figure 2 shows SEM and FM images of both surfaces with their respective SIPN. A fluorescent ring pattern, characteristic of surfaces that were functionalized in the pore,22,23 was observed when the SIPN are incubated. No fluorescence was detected in the surfaces after PNIPAM nanogels were incubated in the same conditions (data not shown). By using the immobilization conditions depicted above, we observed that both SIPN can be successfully immobilized onto the surfaces. In addition, since the nanogels are sensitive to both temperature and pH, we studied the role of the two external conditions on the effectiveness of the immobilization. For that purpose, we analyzed the intensity of the fluorescence exhibited in the pores which is directly related to the amount of nanogel immobilized depending on both the temperature and pH employed for the immobilization. The first experiments were conducted using similar conditions but using two different incubation temperatures, that is, 25 and 45 °C. As can be appreciated in Figure 3, independently of the surface charge of the nanogel, at 25 °C, the fluorescence intensity did not increase after 1 h. After this incubation time E


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be sure that this desorption was not caused by a natural migration of the nanogels toward the aqueous media, a comparative experiment incubating and submerging the SIPN+ in HEPES buffer at pH 6 was performed. The fluorescent values obtained were practically the same as before incubation, evidencing the strong interactions between the SIPN and the surface. Finally, for comparative purposes, NS were incubated with SIPN− at pH 6 and 10. In this range of pH, AMPS is always charged and can be thus effectively immobilized onto the pore surfaces independently of the pH employed. Moreover, rinsing at both pHs did not result in a decrease of the intensity, demonstrating the solid electrostatic interactions. A whole scheme of the pH study for both SIPN+ and SIPN− is depicted in Figure 4. Figure 3. Fluorescence intensity of both negatively and positively charged SIPN when incubated at different times (5 min to 2 h) either at 25 °C or at 45 °C.

and up to 2 h, we reached an equilibrium state in which either no more amino/carboxylic acid groups are available anymore or no additional nanogels are able to reach the functional groups probably due to steric hindrance. In addition, the 2 h incubation time will make sure that the pores of the surfaces are filled with the solution of the nanogels, since there is a transition from the Cassie−Baxter state (where bubbles of air remain trapped inside the pores) to the Wenzel state (where the surface is fully wetted) that should take approximately 1 h. Particularly interesting are the results obtained upon immobilization experiments carried at 45 °C (above Tcp of nanogels) up to 2 h. In this case, a remarkably increase of the fluorescence intensity can be observed in comparison with the observed at 25 °C. This behavior can be explained taking into account the structure of the SIPN. In the one hand, the core of the SIPN, containing thermoresponsive PNIPAm chains, collapses at 45 °C, leaving a higher amount of polyelectrolyte chains in contact with the solvent. This increases the average electrostatic charge of the SIPN, as demonstrated by zeta potential measurements. In principle, an increase of the electrostatic charge density favors the immobilization of a higher amount of nanogel. As has been also depicted below, controls with PNIPAM-dPG nanogels at 45 °C were carried out and no fluorescence was observed under the same experimental conditions. The influence of the pH on the immobilization of the SIPN was also followed by measuring the fluorescence intensity of the films. In the case of SIPN+, prepared using PDMAEMA, they exhibit a positive charge below the pKa (7.4−7.8). SIPN+, 0.5 mg mL−1, was incubated onto AS in HEPES 10 mM solutions at different pH (6 or 10) for 1 h. Fluorescence was only observed when incubated at pH 6, not when incubated at pH 10. This evidenced that SIPN+ nanogels are practically deprotonated at basic pH and, since there is no electrostatic interaction between the SIPN+ and the AS, no adsorption is observed. In contrast, at pH 6, the SPIN+ is electrostatically attached into the cavities of the AS surface functionalized with carboxyl groups. Once pH dependence was proved, it was explored the possibility of desorbing SIPN+ from the AS by washing at pH 10, after having been immobilized the SIPN+ at pH 6. Whereas rinsing at pH 10 immediately after incubation of nanogels at pH 6 practically did not show any decrease of the fluorescence intensity, a high decrease was observed after these samples were immersed overnight in a HEPES solution at pH 10, indicating the removal of the nanogels from the pores. To

Figure 4. Fluorescence intensity values (grayscale) of SIPN+ and SIPN− incubated for 1 h onto either NS or AS varying both the incubation pH and the pH of the buffer solution employed to rinse the porous surfaces.

The above-described experiments based on the measurements of the fluorescence constitute the first insight on the selective immobilization of the gels within the pore. However, these experiments cannot conclude whether some nonspecific adsorption is simultaneously occurring.28 To further analyze the selective adsorption of the nanogels inside the pores,29,30 AFM imaging of the surfaces decorated with the SIPN was performed. AFM is an interesting tool to determine the regions in which the nanogel has been immobilized. AFM measurements of SIPN+ immobilized onto the AS porous surfaces are shown in Figure 5. The first image and cross-sectional profile (Figure 5a) depict the well-defined arrangement of the pores, and upon analysis of the top surface it did not appear to immobilize SIPN +. However, the analysis of the interior of the pores could not be carried out directly and required a peeling of the top surface. Upon peeling (Figure 5b), we removed the top surface and the new topography, also known as pin-cushion structure, permits the analysis of the interior of the cavities. As a result of the peeling, we observed pores with sizes of ∼5 μm also exhibiting a hexagonal array. Further analysis at the nanometer scale on each pore proves that the nanogels are able to penetrate inside of the holes and establish electrostatic interactions that could not be disturbed upon rinsing (Figure 5c). The size of the nanogels measured by AFM inside the pores varied from 40 up to 150 nm with an average value of around 70 nm. According to the nanogels’ sizes measured in solution, these spherical objects can only correspond to the dried nanogels. However, in addition to individual nanogels, some larger aggregates can be appreciated in AFM, probably, because of the tendency of nanogels to aggregate. The same procedure was attempted with F


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Figure 5. Illustrative cartoon (left), AFM height images and cross-sectional profiles (center), and 3D images (right) of (a) the porous film obtained directly after formation and incubation with SIPN+ (20 μm × 20 μm), (b) the previous film peeled (20 μm × 20 μm), and (c) zoom within the pores of the peeled film (1.5 μm × 1.5 μm).

Figure 6. Controlled BSA-Rh protein delivery from (a) AS with SIPN+ and (b) NS with SIPN−. Charged SIPN were incubated for 2 h. Immersion of samples to release the protein was performed for 1 h. Insets after immersion correspond to bright field images from the same area.

protein. In addition to the fluorescence images, Figure 7 shows the fluorescence intensity values that permit one to better quantify this process. It can be seen that the intensities corresponding to the BSA-Rh of the surface slightly decrease, probably caused by some diffusion of the protein to the medium. However, these values remain practically constant because the nanogels have not collapsed. On the contrary, after incubation at 45 °C, the fluorescence intensity values decreased to 0, proving a complete release of the BSA to the medium.

NS. Unfortunately, when peeling was done in these surfaces, the whole material was detached, making it impossible to have a peeled surface. Controlled Protein Release from the Porous Honeycomb Films. Finally, as proof of concept of the applicability of these stimuli-responsive surfaces to deliver encapsulated biomacromolecules, we evaluated their applicability for the release of BSA as model protein. To test the behavior of the SIPN, BSA loaded SIPN were incubated onto the surfaces at pH 6 for 2 h at 25 °C and immersed afterward in a 10 mM HEPES buffer at either 25 or 45 °C for 1 h. As control, PNIPAM-dPG containing BSA was also incubated in the same conditions. In this case, no fluorescence was observed at 25 or 45 °C (data not shown), proving that there does not exist any interaction between BSA and the surfaces. As depicted in Figure 6, the surfaces treated at 25 °C still maintain the fluorescent pattern, because the protein is still encapsulated inside of the nanogels. However, when the treatment is done at 45 °C, no fluorescence was observed on the surfaces. At this temperature, the SIPN collapses, releasing the fluorescent

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CONCLUSIONS We have synthesized and immobilized selectively PNIPAMdPG nanogels semi-interpenetrated with polyelectrolytes on honeycomb polymer surfaces. The SIPN responsiveness to temperature or pH allowed the control of their adsorption− desorption kinetics as well as their collapse−swelling transition. As a proof of concept, we have encapsulated a fluorescent protein (BSA-Rh) inside of the SIPN that was later released upon a thermal trigger. After immobilization of the nanogels onto the polymeric surfaces, a controlled release of the protein G


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Langmuir

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Figure 7. Left: Fluorescence intensity values of AS with SIPN+ charged with BSA-Rh after incubation for 2 h, after subsequent immersion at 25 °C for 1 h, and after immersion at 45 °C for 1 h. Right: Fluorescence intensity values of NS with SIPN− charged with BSA-Rh after incubation for 2 h, after immersion at 25 °C for 1 h, and after immersion at 45 °C for 1 h.

was performed only when the temperature was above the Tcp of the nanogels. These polymeric nanogel platforms may serve not only as functional supports but also as macromolecular carriers of active molecules (such as imaging probes, proteins, drugs, or genes), allowing a specific on-demand release.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b04166. Particle size distribution for SIPN+ and SIPN− determined by DLS (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the MINECO (Projects MAT2010-17016 and MAT2013-47902-C2-1-R (.J.R.H)). A.M.-B. gratefully acknowledges the MINECO for her Ramon y Cajal. A.S.d.L. thanks the Ministerio de Educación for his FPU predoctoral fellowship and DAAD for his short-term scholarship for research stays (A/13/71498). M.C. gratefully acknowledges financial support from the Bundesministerium für Bildung und Forschung (BMBF) through the NanoMatFutur award (13N12561), the Helmholtz Virtual Institute, Multifunctional Biomaterials for Medicine, and the Freie Universität Focus Area Nanoscale. M.M. acknowledges financial support from the Alexander von Humboldt Foundation.

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REFERENCES

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Immobilization of Stimuli-Responsive Nanogels onto Honeycomb

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