Nanostructured Thermal Responsive Materials Synthesized by Soft

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Nanostructured thermal responsive materials synthesized by soft templating. Cedric Vancaeyzeele, Florian Olivier, Gwendoline Petroffe, Sebastien Peralta, and Frederic Vidal ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b00028 • Publication Date (Web): 17 Mar 2017 Downloaded from http://pubs.acs.org on March 21, 2017

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ACS Applied Materials & Interfaces

Nanostructured Thermal Responsive Materials Synthesized by Soft Templating. Cedric Vancaeyzeele*, Florian Olivier†, Gwendoline Petroffe, Sebastien Peralta, Frederic Vidal. Laboratoire de Physicochimie des Polymères et des Interfaces (LPPI – EA 2528), I-Mat, Université de Cergy-Pontoise, 5 mail Gay-Lussac, 95031 Cergy-Pontoise, France.

KEYWORDS. poly(N-isopropylacrylamide), poly(tert-butyl methacrylate), poly(butyl acrylate), controlled release, bicontinuous microemulsion, soft template, amphiphilic material, surfmer.

ABSTRACT. We capitalized herein the inherent tortuosity of bicontinuous microemulsion to conceive nanostructured drug delivery devices. First, we show that it is possible to synthesize bicontinuous materials with continuous hydrophilic domains of PNIPAM network entangled with continuous hydrophobic polymer domains, dual phase continuity being imposed by the bicontinuous microemulsions used as soft template. A particular attention is paid to the microemulsion formulations using a surfmer to preserve the one-to-one replication of the bicontinuous nanostructure after polymerization. These materials keep a volume phase transition with temperature that allows considering them as drug carrier for controlled release. PNIPAM, which plays the role of the active ingredient reservoir, is confined of in the bicontinuous structure. As expected, PNIPAM enclosure limits the surface area in contact with the releasing

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aqueous solution and thus slowed down desorption of aspirin, used as model drug. The hydrophobic polymers play the role of in situ created transport barriers without hindering it since all the loaded aspirin in this bicontinuous structure remains still available.

1. INTRODUCTION. Polymeric systems for controlled drug release are designed to deliver drugs at determined rate over defined periods of time so as to overcome the defects of conventional drug formulation. These defects mainly stand in the initial burst release and a lack in the control of delivery area or temporal modulation of deliverance.1 Hydrogels, that are hydrophilic polymer networks, have been used extensively for the development of drug delivery systems. A hydrogel can swell in polar solvent, such as water, while maintaining the structure.2 It can protect the drug from immune system, enzymes or low pH. It can also control drug release by changing the gel structure in response to a stimulus, such as temperature. Among temperature-sensitive polymers, poly(N-isopropylacrylamide) (PNIPAM) is probably the most extensively studied because of its lower critical solution temperature (LCST) in the range of 32°C, close to the body temperature.3 However, the scope of hydrogel applications is often harshly restricted by their mechanical behavior because most hydrogels are brittle and do not exhibit high stretchability.4 In the purpose of obtaining thermal sensitive hydrogel with reinforced mechanical properties compared to PNIPAM, Reddy et al. synthesized semi-interpenetrating polymer networks (SIPN) in dimethylacetamide (DMAc). They used segmented polyurethane urea (SPUU) and a terpolymer network composed of N-isopropylacrylamide (NIPAM), acrylic acid (AA) and butylmethacrylate (BMA) cross-linked with N,N’-methylenebisacrylamide (MBAM).5 These SIPNs were used for


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comparative drug release studies as function of the formulation at different pH values and at different temperatures to determine the environmental sensitivity of these gels. However, designing of biomaterials using organic solvents may be subjected to controversy. Hence, microemulsion is a tricky but efficient method to produce a soft template for the synthesis of structured materials via a solvent-free process.6 The mixture of hydrophilic and hydrophobic phases is stabilized by surfactants and the corresponding microemulsion is usually optically isotropic and thermodynamically stable. Microemulsion can exhibit a rich diversity of structures among which bicontinuous structure is composed of entangled hydrophobic and hydrophilic domains.7 Even if it is not straightforward, the microemulsion can be turn into a material after polymerization if at least one of the phases contains monomers.8 For instance, Sakata et al. recently developed continuous porous PNIPAM gels prepared by radical polymerization in the water phase of a bicontinuous microemulsion (BME) and with toluene as organic phase.9 The resulting porous PNIPAM gels demonstrated good thermoresponsive behavior and drug releasing rates outperforming those of homogeneous PNIPAM gels. The objective of this paper is then to capitalize on both ideas, i.e. on the inherent tortuosity of such BME based gels and on the improved mechanical propertied afforded by the polymer association through bicontinuous macromolecular architecture to conceive drug delivery device. It should slow down the releasing without hindering it, and thus especially avoids the burst effect that can be observed when delivering a medicine. Here, PNIPAM, which will play the role of the active ingredient reservoir, is confined in the hydrophilic phase of the BME based bicontinuous structure. The hydrophobic phase, on its side, is either composed of poly(tert-butyl methacrylate) (PtBMA) or poly(butyl acrylate) (PBA). As Poly (butyl meth(acrylate)s) cytotoxicity and biocompatibility have already been reported in the literature. 10,11,12,13,14, PtBMA and PBA have


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been considered as potential hydrophobic building blocks for the synthesis of drug release. PtBMA has a glass transition temperature (Tg) at 107 °C and a modulus at room temperature of 2.4 GPa. It should provide rigidity to the material. PBA, that is intended for replacing the former polymer in the hydrophobic phase to bring flexibility to the material, has a Tg at -43 °C and a room temperature modulus of 11 MPa. Here, the materials are made from BME, in which the free-radical polymerization and crosslinking of monomers are photochemically initiated. The surfactant used to stabilize the BME is Brij 35, polyoxyethylene (23) lauryl ether, of which the terminal alcohol function is beforehand modified with 2-isocyanatoethyl methacrylate. A methacrylate function is thus grafted onto the surfactant to give a surfmer.15 The use of surfmer is reported in the literature as favorable for preserving the BME initial structure in the material.16 In addition, for the encapsulation and release of the drug, the material structure must not leak out any chemical, as it would the case of unreactive surfactant. Finally, once the conception of the materials and their characterizations studied, a particular attention is focused on effect of the confinement of the PNIPAM inside the BC structure over the release of aspirin, used as model drug.

2. EXPERIMENTAL SECTION 2.1.

Chemicals.

N-isopropylacrylamide (NIPAM, 97%), N,N-methylenebisacrylamide (MBA, 99%), ethylene glycol dimethacrylate (EGDMA, 98%), 2-isocyanatoethyl methacrylate (ICEMA, 98%) dibutyltin dilaurate (DBTDL, 95%), acetylsalicylic acid (ASA, 99%) and polyoxyethylene (n = 23) lauryl ether (Brij35, 99%) were provided by Sigma-Aldrich, and were used as received.


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Butyl acrylate (BA) and tert-Butyl methacrylate (tBMA, Sigma-Aldrich) were distilled before use. 2-hydroxy-2-methylpropiophenone (Darocur 1173, D1173, Ciba), 1-[4-(2-Hydroxyethoxy)phenyl]-2-hydroxy-2-methyl-1-propane-1-one (Irgacure 2959, I2959, 98%, Sigma-Aldrich), tetrahydrofurane (THF, VWR), dichloromethane (CH2Cl2, VWR) were used as received. Water was deionized through a MilliQ purification system before use.

2.2.

Instrumentation.

Photopolymerization for the synthesis of single networks and BC materials were performed under UV curing lamp (Primarc UV Technology, Minicure, Mercury vapor Lamp, 100 W.cm-1). FTIR spectroscopy was performed on a Bruker spectrometer (Equinox 55) by averaging 16 consecutive scans with a resolution of 4 cm-1. The rates of the network formation in the bulk were followed, in the near infrared (NIR) region (6250-6100 cm-1) by monitoring in real time the disappearance of the H–C=C overtone absorption bands at 6190 cm-1 for N-isopropylacrylamide and 6160 cm-1 for butyl acrylate and terbutyl methacrylate. A given peak area is directly proportional to the reagent concentration (the Beer–Lambert law has been verified), thus the conversion-time profile can be easily derived from the spectra recorded as a function of time. The conversion of reactive bonds can be calculated as p= 1-(At/A0) from the absorbance values, where the symbols have the usual meaning and the subscripts 0 and t denote reaction times, i.e. the time of exposure to the UV curing lamp. The materials were synthesized in a mold made of two glass plates clamped together and sealed with a 1 mm thick Teflon® gasket. The ultraviolet-visible (UV-vis) spectra were recorded in Suprasil® cell (pathlength 10 mm, Hellma) on a V570 spectrometer (Jasco) in transmission mode with 2 nm bandwidth at 400 nm min-1.


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H and 13C NMR spectra were recorded with a Bruker DPX250 spectrometer in CDCl3 as solvent

and reference. All chemical shift values are given in ppm. Rheological measurements were performed with an Anton Paar Physica MCR 301 rheometer equipped with CTD 450 temperature control device and a cone−plate geometry (cone: diameter 25 mm, angle 2°; plate: polymerization system made from glass coupled with U.V Source Omnicure). A 0.5% deformation was imposed at 1 Hz. Storage modulus (G′) and loss modulus (G″) were recorded as a function of time. The gel time was determined at the intersection between storage and loss modulus curves. The solution of precursors was put in the geometry and measurement began immediately with U.V exposure (Hg vapor lamp, 19 W cm-2) at 25 °C. Data were acquired with Rheoplus v3.40 software. Surface tension measurements were performed using the Du Noüy ring detachment method (Kruss K6 tensiometer). The planar and spherical ring was placed parallel to the air/aqueous interface (pH 7). Between the surface tension analyses, the ring was cleaned by rinsing it with milli-Q water. Temperature was kept at 25 °C during all experiments. Microemulsions and materials were imaged by confocal laser scanning microscopy (LSM 710, Zeiss, Germany) using the ZEN2010 software. 0.3 wt% 9, 10-Diphenylanthracene (DPA, Acros, 99%) and 0.04 wt% rhodamine B (RB, Aldrich) were dissolved before emulsification in the organic and aqueous phases, respectively. For the fluorescence contrast method, excitation wavelengths were 405 nm (30 mW, emission window from 420 to 460 nm) and 561 nm (20 mW, emission window from 570 to 640 nm). These measurements were performed with a planapochromatic objective (63x/1.40 oil immersion). Each line was scanned and averaged four


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times with a field resolution of 1024 x1024 pixels and 16 bit dynamic range. The selected images are obtained by focusing in the bulk of the samples Differential scan calorimetry (DSC) was performed on a DSC Q100 model (TA Instruments). The thermograms were recorded at 20°C min-1 heating rate between -90°C and 150°C. In between the first and second scan the sample temperature decreases at -10°C/min to favor any possible crystallization. Thermal transitions were detected on the second scan. Static contact angles were determined with a KRUSS® DSA10 MK2 goniometer. 15 µL of ultrapure water drop were deposited on a dried surface and the reported values are means of at least three measurements. The atomic force microscopy (AFM) experiments were performed in Peak Force QNM mode with a Dimension ICON microscope from Bruker. Measurements were carried out in air at room temperature with a tip model ScanAsyst (k = 0.4 N/m, Bruker) on the surface of dried samples and then on surface of the same fully hydrated samples. The deflection, the stiffness constant and the probe shape were calibrated according to the manufacturer procedure. All images were made with the same probe with the same peak force. Images were analyzed with NanoScope Analysis 1.5. The soluble fractions (SF) contained in the dried materials were extracted in a Soxhlet® for 72 h with tetrahydrofuran (THF). After extraction, the sample was dried again under vacuum at 40°C until constant weight and then weighed. The soluble fraction was determined as the weight percentage: SF (%) =

(W0 − W E ) × 100 , where W and W are the sample weights before and after 0 E W0


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extraction, respectively. The soluble fractions were then analyzed by 1H NMR using the Bruker Avance DPX250. Water uptakes (WU) of the materials were determined by immersion in deionized water in isotherm for 24h. The sample weight was measured (w1). Then the material was dried until constant weight (w2). The water uptake was calculated as (%) =

( − ) × 100.

For drug loading experiments, the polymer sample was dried and weighted (wpolymer) after Soxhlet extraction for 72h with THF. It was immersed in acetylsalicylic acid (ASA) solution in ethanol (150 g/L) for 24h at 25°C. Then, the sample surfaces were wiped and this loaded sample was dried until constant weight (wpolymer+drug). The weight ratio of Acetylsalicylic acid (ASA) absorbed is calculated as % = (

!

− )⁄ × 100

Drug releasing from 0.2 g of material was monitored out in 200 mL deionized water at 25°C, 37°C or 45°C. The cumulative molar concentration of ASA in water was determined by UV titration at 275 nm using external standard calibration curve with Abs(275 nm) = 218.02 [ASA]. The cumulative weight percentage of ASA extracted from the material was calculated from this concentration and the drug loading percentage.

2.3.

Surfmer synthesis from Brij35®.

10 g (8.35 mmol) of dried Brij 35 are dissolved under magnetic stirring in 35 mL dichloromethane, previously distilled and dried on calcium hydride, in a 100 mL 2-neck roundbottom flask connected to a condenser. The solution is purged under argon before addition of


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105 mg dibutyltin dilaurate (0.17 mmol; 1 mol % with respect to 2-isocyanatoethyl methacrylate; 98.5 µL). Then 2.6 g of 2-isocyanatoethyl methacrylate (ICEMA – 16.7 mmol; 2.36 mL) are added dropwise and stirred for 3h at 25°C. The product is washed by precipitation in diethyl ether, dried and stored under vacuum. 1H NMR in CDCl3: 0.75 (t, CH3 al), 1.18 (m, CH2), 1.85 (s, CH3- C=C), 3.39 (m, CH2(al)-O), 3.57 (m, CH2-CH2-O), 3.64 (m, CH2-CH2-OCO), 4.14 (m, CH2OCO), 5.5 (1H, s, CH2=C) and 6.01 ppm (1H, s, CH2=C). 13C NMR in CDCl3, δ (ppm) = 158.3 ppm (C=O, urethane), 166.8 ppm (C=O, methacrylate). FT-IR: υ 1716 cm-1: C=Oester, υ 1096 cm1

O=C-O-Rester, υ 1635 cm-1: C=C and no υ 2270 cm-1: NCO

2.4.

Single network synthesis.

Poly(N-isopropyl acrylamide) (PNIPAM) single network was prepared by dissolving 0.3 g Nisopropyl acrylamide and 0.02 g N,N'-methylene bisacrylamide (MBA) in 1.68 g deionized water. 0.04 g Irgacure 2959 (I2959) were added to the solution degased under argon for 10 min and poured into a mold made of two glass plates clamped together and sealed with a 1 mm thick Teflon® gasket. The solution was exposed under UV for 30 s. A white and soft polymer gel was obtained. Poly(tert-Butyl methacrylate) (PtBMA) single network was synthesized from 0.04 g Darocur 1173 (D1173) dissolved in 1.9 g tert-Butyl methacrylate (tBMA) and 0.1 g ethylene glycol dimethacrylate (EGDMA). The solution was degassed under argon for 10 min and poured into the previously described mold. Polymerization took place for 7 min under UV. A transparent and solid polymer network was obtained. Poly(butyl acrylate) single network was synthesized in the


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same condition, except that the monomer was butyl acrylate (BA) instead of (tBMA) and the UV exposure last 30 s.

2.5.

Polymerization in microemulsion.

The microemulsions structure depends on the aqueous phase, surfactant and organic phase proportions. By analogy with a previous study,17 the Фaq/Surfactant/Фorg weigh percentages are set at 46/38.6/15.4. So, 0.772 g Brij 35surf were dissolved at 40°C in the organic phase which was composed of 0.293 g tBMA and 15 mg EGDMA degassed under argon. Emulsification was carried out by the addition under mechanical stirring of the aqueous phase composed of 0.138 g NIPAM and 9 mg MBA previously dissolved in 0.772 g deionized water degassed under argon. For the radical polymerization in microemulsion either 18 mg I2959 were added to the aqueous phase or 6 mg D1173 to the organic phase at room temperature before emulsification. The microemulsion was a highly viscous gel that was poured into the previously described mold and exposed to UV during 6 min for photopolymerization. Transparent materials were obtained. After synthesis the materials contained mainly water. They were dried under vacuum until constant weight for any characterization in dry state.

3. RESULTS AND DISCUSSION The aim of the first part of this work is to synthesize bicontinuous materials with continuous hydrophilic domains of PNIPAM network entangled with continuous hydrophobic polymer domains. The hydrophobic polymer will provide mechanical robustness and resistance to erosion


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to the hydrophilic polymer in medical and pharmacological applications.18 Then, the confinement of PNIPAM is expected to interfere on the releasing properties compared with bulk PNIPAM gel. PNIPAM is a thermal sensitive polymer that shows in aqueous solution a reversible transition around 32°C known as the low critical solution temperature (LCST). Below 32°C, the PNIPAM is hydrophilic with chains in extended conformation, and surrounded with water molecules because of hydrogen interactions while above the polymer-polymer hydrophobic interactions predominate over the hydrogen bonds.19 The polymer chains dehydrate and collapse before precipitating. Poly (tert-butyl methacrylate) has a glass transition temperature of 107°C,20 higher than ambient temperature. It was chosen to provide stiffness (E’ = 2400 MPa at RT) and also to reduce the water absorption by the hydrophilic material of the final network by entrapping the hydrophilic network in a rigid skeleton. This polymer network has also been exchanged by another based on poly(butyl acrylate). It is a soft polymer (E’ = 11 MPa at RT) with low Tg at -43°C. Thus, it should hardly limit the volume variation during hydration and dehydration of the hydrophilic partner. The effect of the scaffold rigidity on PNIPAM properties is then further studied. During the materials synthesis, the dual phase continuity21 is imposed by the bicontinuous microemulsions used as template.6 The microemulsions structure at fixed temperature is governed by the proportion of aqueous phase, surfactant and organic phase. By analogy with a previous study17 in which Brij 35 was also used as surfactant, the Фaq/Surfactant/Фorg weigh percentage are set at 46/38.6/15.4. As these materials are specially designed to get a gyroid-like structure in which PNIPAM is confined to tune the releasing properties. Hence, the synthesis and characterization of hydrophobic and hydrophilic single networks is studied beforehand. A particular attention will be paid to get no extractable chemicals. Thus, the surfactant has been


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modified with methacrylate function so as to chemically graft it inside the material. This reactive surfactant is noted as Brij 35Surf and has been defined according to the literature as a surfmer.15

3.1. Synthesis of the surfmer Brij 35Surf The surfmer synthesis consists in the addition reaction of the alcohol function of Brij 35 with the isocyanate of 2-isocyanatoethyl methacrylate (ICEMA) to form a urethane bond (Figure 1).

Figure 1 : Schema of the synthesis of the surfmer Brij 35Surf

Brij 35 (1 mol equivalent) is dissolved in dried dichloromethane with DBTDL catalyst. ICEMA (2 mol equivalent) is added dropwise and stirred for 3h more at 25°C. The surfmer is purified by precipitation in diethyl ether. The thermal properties of the purified surfmer are reported in figure S1 (Supporting information). The surfmer and the commercial surfactant are both semicrystalline oligomers since they have two thermal transitions in DSC: a glass transition (Tg onset at -54°C and -31°C for the Brij35Surf and Brij 35, respectively) and a melting temperature (Tm at 24°C and 44°C for the Brij35Surf and Brij 35, respectively). The modification of thermal properties can be attributed to the bulky methacrylate group grafted on the polar head of the surfmer. More free volume and less susceptibility to form hydrogen bonds decrease Tg and Tm.


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Similarly, Brij 35 has been reported to have a cloud point above 100°C22 whereas Brij35Surf displays a cloud point at 51°C. This decrease of cloud point witnesses that methacrylate end group reduces the hydrophilicity of polyoxyethylated polar head of the surfmer. The critical micelle concentration (CMC) of Brij 35Surf has been determined by Du Noüy ring method and compared with that of the commercial Brij 35 (Figure S2). The modification of the hydroxyl group on polar head of commercial Brij 35 with a less polar group reduces the CMC from around 50 to 25 µmol/L for Brij 35Surf. The enhancement of hydrophobicity is further confirmed by a lower surface tension which is lower on average of 2 mN/m (46 mN/m for Brij 35 and 44 mN/m for Brij 35Surf at 100 µmol/L for instance). First as a blank, the hydrophilic network is synthesized by free-radical photopolymerization initiated by 2 wt% (in regards to the total monomer) Irgacure® 2959 (I2959) of 15 wt% NIPAM with 1 wt% MBA as cross-linker in deionized water. This proportion is kept constant all along this study. The rate of this single network formation was followed by monitoring the disappearance of the H-C=C overtone absorption band at 6180 cm-1 after every 30 s of UV (100 W cm-1) exposure (Figure 2). This band totally disappears within the first 30 s of UV exposure. The conversion is fast and complete. Soxhlet extraction in THF for 3 days of this sample gives soluble fraction of 3 wt% that was identified by 1H NMR as linear PNIPAM. Obviously, the PNIPAM single network is correctly cross-linked.


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PtBMA

0s

0 min Absorbance

Absorbance

PNIPAM

30s

6220

6170

6120

6 min 7 to 9 min

6250

6200 6150 Wave number(cm-1)

Wave number (cm-1)

PNIPAM PBA PtBMA

Conversion (%)

80

1 min 2 min

6100

100

PBA

0 min Absorbance

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60 40 20

6250

6200

6150

Wave number

(cm-1)

6100

0 0

1

2

3

4

5

6

7

8

Time(min) Figure 2: NIR-FTIR spectrum and conversion of the acrylamide, methacrylate or acrylate functions during the formation of the PNIPAM, PtBMA or PBA single networks, respectively.

Second, the poly(tert-Butyl methacrylate) (PtBMA) single network is synthesized in bulk by free radical polymerization of 95 wt% tBMA and 5 wt% EGDMA initiated by Darocur® 1173 (D1173) under UV. The single network formation is followed by the disappearance of H-C=C overtone absorption band at 6160 cm-1 (Figure 2). The conversion is slower than that of the PNIPAM single network. Indeed, only 50 mol% of the methacrylate functions have reacted after 6 min. Next, the conversion jumps up to 90 mol% during the following minute of UV exposure and do not increase anymore afterwards. This jump is classically attributed to Trommsdorff-Norrish effect that occurs during the polymerization in bulk of high Tg polymethacrylate.23,24 The incomplete conversion (90%) of methacrylate functions witnesses that the polymerization is


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diffusion-limited because of gel effect.25 Hence the PtBMA network synthesis can be considered as optimal after 7 min. It was then extracted with Soxhlet® for 3 days in THF. The soluble fraction is 3 wt% of linear PtBMA as determined by 1H NMR which indicates that this network is correctly formed. Somehow the 7 % discrepancy is either probably due to unreacted monomer evaporation or residual unsaturations on pendant groups. Third, the poly(butyl acrylate) (PBA) single network is synthesized in bulk by free-radical photopolymerization of 95 wt% BA and 5 wt% EGDMA. The reaction is initiated by Darocur® 1173 under UV. The NIR FTIR monitoring gives a conversion curve in which the acrylate function have completely reacted within 2 min, which is in agreement with higher propagation rate for acrylate compared to methacrylate usually reported in the literature.26 Finally Soxhlet extractions in THF on this sample give soluble fractions of 5 wt%, confirming the correct polymer cross-linking. From these syntheses, one can conclude that those three networks can be included in the following bicontinuous materials since they are correctly formed. PNIPAM will be synthesized in the aqueous phase of the microemulsion and PtBMA or PBA in the organic phase.

3.2. Microemulsion formulation and material synthesis The microemulsion is composed of 46 wt% aqueous phase, 15.4 wt% organic phase and 38.6 wt% Brij 35Surf as surfmer.17 Each phase is composed of the precursor of single networks that were previously described (Table 1).


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Phase

Aqueous

Organic Brij 35Surf

Phase Proportion (wt%)

46

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Component in the phase

Component proportion in each phase (%)

NIPAM

15

MBA

1

Water

84

tBMA (or BA)

95

EGDMA

5

-

-

15.4 38.6

Table 1: Composition of the microemulsions

The free radical polymerization initiation can be attempted either from the organic phase, or the aqueous phase or both phases of the microemulsion template. The effect of the initiation locus of the radical polymerization in the microemulsion template on the rate of monomer conversion and on the final material morphology was checked (supporting information). As no changes are observed, only one photoinitiator is sufficient. D1173 is selected for the continuation of this study only because of practical reason: it is liquid and dilutes quickly in the organic phase Фorg. 2wt% of D1173 in regards to total monomers and cross-linkers are dissolved in the organic phase before emulsification. The microemulsions obtained are viscous transparent liquid. They are molded and the polymerizations are initiated under UV as previously described for the single networks. With these studied bicontinuous microemulsion formulations, all the features required to preserve the one-to-one replication of the bicontinuous nanostructure after polymerization are gathered according to current approaches described in the literature.27 (1-) The addition of crosslinkers reduces further the mobility of the polymer so that cross-linked regions do not reorganize. Here polymer network are synthesized in aqueous and organic phases. As the


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curvature of the surfactant film governs the phase structure behavior, (2-) a surfactant of high molecular weight (Brij 35Surf) is used for the emulsification28 of (3-) highly viscous bicontinuous phases structure (average viscosity

1.5 Pa.s), featuring slower rearrangement

dynamics. (4-) Finally, to retain this preferred curvature and slower surfactant rearrangement dynamics in the course of polymerization, a surfmer Brij 35Surf has been synthesized from Brij35. 29,30 The materials obtained are named I and II when monomers are respectively tBMA or BA. The rate of material formations were followed in the near infrared (NIR) region (6250–6100 cm-1) by monitoring the disappearance of the H-C=C overtone absorption band at 6170 cm-1 after every 30s of UV exposure (Figure 3). With this technique the acrylamide double bound cannot be distinguished neither from methacrylate nor acrylate double bounds. So it gives only a picture of the overall function H-C=C conversion.

Figure 3: Conversion of the acrylamide and methacrylate functions during the formation of I as well as the conversion of the acrylamide and acrylate functions during the formation of material II. Photos of the resulting materials after polymerization.

FTIR monitoring of materials I and II formations show that the reactive functions H-C=C have completely disappeared within 2 min. The conversion yield of 100% is reached with a difference


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in rate of polymerization that follows the classical trend of the monomer reactivity: acrylates, more reactive than methacrylate. The materials formed are soft and transparent hydrogels. To evaluate the cross-linking effectiveness, Soxhlet extractions are performed for 72h in THF, a good solvent of the compound from both phases and the surfmer. Soluble fractions of the dried samples are equal to 8wt%, which demonstrates that the polymer is correctly cross-linked. 1H NMR analyses of these factions show mixture of surfmer and polymer of the organic phases. These measurements confirm that the networks are correctly formed and that most of the surfmer molecules are grafted on them. Thus, the next step consists in observing the morphology of the hydrated materials by confocal laser scanning microscopy (CLSM) to determine if the structure of the microemulsion is an effective template to get nanostructured materials. In that purpose, Фorg is selectively stained blue with 9,10- diphenylanthracene (DPA) and Фaq stained red with rhodamine B (RB) before emulsification. Representative CLSM images of the fluorescence of these two phases in the microemulsion and in the materials I and II are presented in Figure 4. The selected images are obtained by focusing in the bulk of the samples.


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Fluorescence

Fluorescence

in Фaq (RB)

in Фorg (DPA)

sample

microemulsion

I (PtBMA)

materials

microemulsion

II (PBA)

material

Figure 4: CLSM image of I and II: microemulsion templates and the materials after photopolymerization. Selective staining of the two phase are done: Фaq stained red with rhodamine B and Фorg stained blue with 9,10diphenylanthracene.


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In the microscopy images of the microemulsions (soft template of materials I and II), labyrinthlike nanostructure can be distinguished in the red and blue CLMS fluorescence images. The two dyes, strictly soluble in one of the two phases and emitting in very different wavelength range, are almost co-localized, down to the resolution of this optical microscope. Thus the aqueous and the organic phase appear continuous. The CLMS images of the materials I and II show the same kind of co-continuous structure in Фaq and Фorg. The BC structure is kept after polymerization since it gives CLSM images identical to those of the microemulsions. So the templates structure seems to be preserved after polymerization; the BC microemulsions generate BC materials. It is noteworthy that these CLSM images are identical to those previously observed on BC microemulsions and materials17 of which the structures have been confirmed by SAXS and WAXS.31 It was observed that the long-range ordered cubic bicontinuous structure was slightly destabilized during polymerization to give another less-defined bicontinuous structure. In consequence two bicontinuous materials in which a PNIPAM network is confined are synthesized. Their essential difference is the nature of their hydrophobic component. The material I is based on PtBMA and the material II on PBA. One of the objectives of this paper is to improve mechanical properties by the polymer association through bicontinuous macromolecular architecture to conceive drug delivery device. PNIPAM single hydrogel, material I and II rheological properties were measured in situ during their synthesis (Figure 5).


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Figure 5 : Storage modulus G′ (full) and loss modulus G’’ (empty) as a function of the UV irradiation time for the synthesis of () material I, () material II and () PNIPAM single hydrogel.

First the rheological characterization in Figure 5 confirms the fast formation of the material II and PNIPAM single network with a gel time below 30s compared to that of the material I (gel time around 60s). After 180s of UV radiation, moduli of the three samples are stable and representative of the final materials. The storage moduli G’ are equal to 10, 175 and 410 kPA for PNIPAM single hydrogel, material II and material I, respectively. The bicontinuous materials have a storage modulus at least one order of magnitude higher than that of PNIPAM. As expected the storage modulus of material I based on the stiff PtBMA is higher than that of the material II based on the soft hydrophobic PBA. Finally, the PNIPAM association with PBA or PtBMA through bicontinuous macromolecular architecture allows improving the mechanical properties of hydrogels.


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In the next section, materials bulk properties will be examined given the well-known absorption/release properties of PNIPAM. However, the surface properties are studied beforehand to give the extent of the influence of the hydrophobic partner on the materials properties.

3.3. Characterization of the materials I and II. 3.3.1. Surface Properties. As the bicontinuous structures of the materials have been checked, the presence of the two-phase domains may influence the surface properties.32 At first the BC materials were completely dried and then their wettability was examined in measuring contact angle formed by a water droplet on their surface (Table 2). As blanks, the single networks have also been studied. samples

Angles

PtBMA

77.0 ± 7.0°

I

38.3 ± 5.0°

PBA

87.0 ± 9.0°

Photo


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II

51.5±5°

Table 2: Contact angles and photos of water droplet on dry surface of PtBMA and PBA single networks and materials I and II. Experiments were done at room temperature.

It was not possible to register any contact angle on PNIPAM. The water droplet absorbs almost instantaneously because PNIPAM is a hydrogel. PtBMA and PBA networks display similar contact angle of around 80-90°, typical of materials displaying a low wettability. Hence, complete wetting is expected for BC materials composed of PNIPAM if this hydrophilic polymer is at the surface. The water droplet on bicontinuous material I makes a reduced angle of 38°, Besides, the water droplets and bicontinuous materials II show a contact angle equal to 51°. The apparent contact angles are in between those observed on the PNIPAM and hydrophobic PtBMA or PBA single networks.33 In order to check if there could be any surface chemical composition variation upon dehydration, materials I (Figure 6) and II (Figure 7) were observed by atomic force microscopy (AFM) in both dry and wet state.


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Figure 6: AFM images of the surface of material I. On the image a, b and c the sample is dried. a’ and b’ the sample is water swollen. a and a’ are topography image, b and b’ adhesion force images and c is modulus image.


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Figure 7: AFM images of the surface of material II. On the image a, b and c the sample is dried. a’ and b’ the sample is water swollen. a and a’ are topography image, b and b’ adhesion force images and c is modulus image.

AFM images were recorded at room temperature on the surface of dried samples and then on surface of the same fully hydrated samples. Representative images are reported in Figure 6 for material I and in Figure 7 for material II. It is worth noting however that the samples are first dried before AFM experiments. So the observed patterns of around 200 nm are quite distorted


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even for the re-hydrated ones and not representative of smaller domain size typical of bicontinuous microemulsion.34 In consequence no domain size estimation is drawn from these images. The dried material I has a fairly low mean roughness (RRMS) of 45 nm that is estimated from the topography image (Figure 6a). Mean adhesion force and mean modulus are respectively calculated from adhesion force image (Figure 6b) and modulus image (Figure 6c). Mean adhesion force is low and equal to 26 nN as expected with a stiff sample, which has a mean modulus of 2210 MPa. In addition, accurate observation of the topography shows flat surface with randomly distributed depressions that exactly correspond to the most polar and soft part of the sample, according respectively to the adhesion force image and modulus image. Silicon AFM tip is hydrophilic, so the most interacting areas appear brighter. Hence the scenario, in which the polar PNIPAM hydrogel contract upon drying (scheme in Figure 8), is assumed. The PtBMA is a rigid and high Tg polymer. It is not sensitive to the volume variation of polar part. The hydrophobic part of the sample I is exposed at the surface and turn it more hydrophobic than the single PNIPAM network. Once the sample I is fully hydrated (24h immersion in water) the surface roughness remains low at 20 nm and the height contrast is smoothed by the water swelling of the hydrophilic domains (Figure 6a’). The same pattern is observed on the adhesion force image (Figure 6b’). The dried material II has a very low roughness, RRMS = 3.6 nm (calculated from Figure 7a). The mean adhesion force (calculated from Figure 7b) and the mean modulus (calculated from Figure 7c) are respectively equal to 50 nN and 158 MPa. These values are in agreement with the sample composition. Indeed, the hydrophobic PBA is a low Tg polymer usually used in paint, coating, caulk, sealant, adhesive formulation for its adhering and film forming properties.35


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There is a correlation between the height (Figure 7a) and modulus (Figure 7c) contrast with a flat surface displaying some dispersed stiff prominent dots after the material has collapsed upon drying. Simultaneously, the adhesion force image Figure 6b shows low adhering spots dispersed on the fairly adhering surface. Given the Tg range difference between the two polymers it is obvious that the adhesion force image is govern by polymer toughness and not by polar interaction between AFM tip and polymer. Indeed, adhesion force must be higher for the PBA rubber (in clear in Figure 7b) than the dried and hard PNIPAM (Tg = 147°C 36, in dark in 7b). In this case, the soft PBA is free to expend while PNIPAM contract upon drying (scheme in 8). So, the hydrophobic PBA part of the sample is exposed mainly at the surface, which is in accordance with higher contact angles compared with material I (Table 2). Once fully hydrated (24h immersion in water), material II shows the same behavior than the sample I: there are reduced contrasts in the topography image (Figure 7a’), with a surface roughness that remains low at 5.2 nm.

I Rigid PtBMA φhydrophobic

φhydrophilic Dry PNIPAM

Dehydration hydration

II Soft PBA Dry PNIPAM

Figure 8: scheme of alternating hydrophilic and hydrophobic phase domains at the surface of BC materials and consequence of depression on their wettability.

In figure 8, the schema shows the two different behaviors expected for BC structure upon dehydration. PtBMA is a stiff polymer and should not change its shape while the PNIPAM is


27


drying whereas PBA is soft and should expend while the PNIPAM collapse. Overall, the surfaces analyses of the BC samples show that PNIPAM and the other polymer are also bicontinuous at the surface. 3.3.2. Volume phase transition and water uptake As previously mentioned, PNIPAM is mainly used as thermoresponsive hydrogel due to its LCST (32°C). It is often called as volume phase transition Temperature (VPTT) in gels.37 When cross-linked, water swollen PNIPAM remains translucent regardless the temperature. So the most practical method to measure the VPTT is the monitoring of the successive weight of water absorbed by the polymer network as the function of the temperature (Figure 9).

800

180

750

160

700

140

a

Water uptake (wt%)

Water uptake by PNIPAM network (wt%)

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650 600 550 500 450

material II

120 100 80 60

400

40

350

20

300

material I

b

0 25

30

35

40

45

50

25

Temperature (°C)

30

35

40

45

50

Temperature (°C)

Figure 9: Water absorption by PNIPAM network (a) and materials I and II (b) as function of temperature.

The two hydrophobic polymer networks, i.e. PtBMA and PBA, display residual water absorption of 1-3% whatever the temperature (data not shown). In Figure 9a, the water uptake of PNIPAM network decreases from 725 wt% at 25°C to 480 wt% at 50°C. The VPTT of PNIPAM corresponds to the slope break at around 37°C, which is slightly higher than the data from the


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literature 32°C.38 So, there is no abrupt reversible change in the PNIPAM network swelling from hydrophilic to hydrophobic but rather a worse capacity to absorb water with the temperature.39,40,41 VPTT of materials I and II were also determined using water absorption as function of the temperature (Figure 9b). At 25°C the ratio of water absorbed is greatly diminished compared to PNIPAM single network i.e 725 wt% for PNIPAM for less that 100wt% for the two materials. It may be due to the weight proportion between the hydrophilic and hydrophobic polymer networks in the material; the hydrophilic part, i.e; the PNIPAM network and the PEO surfmer is only accounting for 59 wt% in the whole material. Indeed, since the content of surfactant is high in the microemulsion, any discussion on the water uptake should take into account the presence of the grafted surfactant in the material. In the same way as PNIPAM, these materials have reduced hydrophilic behaviors when the temperature increases, but contrary to PNIPAM, the water absorption decreases linearly with the temperature from 25°C to 50°C without any slope break. In addition to the thermoresponsive properties of the PNIPAM, PEO interaction with water is less favorable as temperature increases.42 Thus the cumulated water uptake by PNIPAM and PEO versus temperature could be the reason for the lack of break point in the water absorption curves in Figure 9b. The material I has a water uptake (WU) of 95 wt% at 25°C and 80 wt% at 50°C (∆WU = 15wt%) and material II has a WU of 80 wt% at 25°C and 50 wt% at 50°C (∆WU = 30wt%). Despite the absence of break point in the water absorption curves, materials I and II are now considered as drug carrier for controlled release.

3.4. Drug loading and release Acetylsalicylic acid (ASA) was selected as model drug. PNIPAM single network and bicontinuous materials I and II were first THF extracted for 72h in order to take off any chemical


29


from these scaffolds. The drug loading (Figure 10) is carried out in ethanol because it is a good solvent of ASA and PNIPAM. The weight ratio of loaded ASA is calculated from the weight difference on dried samples before and after immersion. 2,5 Normalyzed concentration of Salicylic acid absrobed/ %PNIPAM

70% 60%

% Salicylic acid loading

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50% 40% 30% 20% 10% 0% PNIPAM

Mat I

Mat II

2 1,5 1 0,5 0 PNIPAM

a

Mat I

Mat II

b

Figure 10: (a) Weight percentage of acetylsalicylic acid (ASA) loaded in PNIPAM and materials I and II. (b) Normalized proportion of ASA loaded per weight % of PNIPAM in the materials.

The single PNIPAM hydrogel can absorb up to 59 wt% of ASA whereas the loading is reduced to around 13 wt% in the BC materials (Figure 10a). The result is in agreement with the lower proportion of hydrophilic phase prone to absorb ASA solution of ethanol. The Figure 10b shows the concentration of absorbed ASA per g of PNIPAM. The normalized absorptions are higher for materials I and II than for the single PNIPAM network. It highlights that BC materials still efficiently absorbs ASA and that PEO groups of the surfmer, together with PNIPAM, contribute to ASA absorption. Controlled release of drugs from a scaffold can accelerate the locale therapeutic process and bypass undesired side effects of a drug that are magnified because of over-exposure. In the same time, the drug concentration has to be above a threshold to be effective, all together with a


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relevant dose at the targeted site for an extended period of time.43 The materials I and II are designed to promote thermally regulated release. So, drug release experiments were carried out to display the influence of the material’s nano-structuration on the releasing properties. The experiments were done in pure water over a relatively short period of time (24h). Drug releases from samples were conducted at 25°C and 37°C. The molar concentration of ASA in water was determined by UV at 275 nm according to Beer-Lambert law. The cumulative ASA release can thus be estimated using the drug loading percentage (Figure 11).


31


100

% of cumulative salicylic acid released


100 90 80 70 60 25°C 50 37°C

40

PNIPAM

30 20

a

10 0

90 80 70 60 25°C 50 37°C

40

Mat I

30 20

b

10 0

0

3

6

9

12 15 Time (h)

18

21

0

24

3

6

9

12 15 Time (h)

18

21

24


100 90 80 70 60 50

25°C

40 37°C

30

Mat II

20

c

10 0 0

3

6

9

12 15 Time (h)

18

21

24

100 % of cumulative salicylic acid released

100 % of cumulative salicylic acid released

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90 80 70 60 50

PNIPAM

40

Mat I

30

At 25°C

20

Mat II

d

10 0

90 80 70 60 50

PNIPAM

40 Mat I 30

At 37°C

20

Mat II

e

10 0

0

3

6

9

12 15 Time (h)

18

21

24

0

3

6

9

12 15 Time (h)

18

21

24

Figure 11: Cumulative percentage of acetylsalicylic acid released from PNIPAM (a), material I (b) and II (c) in deionized water at 25°C or 37°C. Cumulative percentage of acetylsalicylic acid released at 25°C (d) or 37°C (e) for the three materials.


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The ASA releasing from PNIPAM and materials I and II have also been monitored at 45°C. In all the case the releasing curves overlap those registered at 37°C (Figure S5). So the experiments run at this temperature are not discussed here. At 25 ° C, the cumulative release of ASA from the PNIPAM single hydrogel (Figure 11a) reaches a plateau after 6 hours and 75% drug are finally released after 24h. The curve at 37 ° C has higher initial rate and approximately 87% of the ASA is extracted after 3 hours, which is the time needed to reach a plateau. These percentages change little and reach 95% after 24 hours. At room temperature, the release profile from I (Figure 11b) and II (Figure 11c) materials, are similar over the first 6 hours with less than 45% of ASA released. After 24 hours, the maximum of acid released into water is up to 90% for the material I and 80% for the material II. Thus the ASA extractions after 24h from the materials I and II are greater than that of PNIPAM (75%). ASA release profiles follow a similar trend for materials I and II, i.e. a relatively slow desorption at 25°C and accelerated at 37. The percentage released after 6h is equal to around 95% and 70%, respectively for materials I and II and the released after 24h from material I is complete whereas it is around 90% from material II. From these released profiles, one can assume that at 25°C the hydrophilic polymer chains interact with ASA through hydrogen bonds whereas at 37°C the hydrophobic behavior of PNIPAM and PEO groups prevails. Polymer chains do not interact anymore with ASA and the drug is expelled altogether with water. The monitoring at 25°C and 37°C, corresponding to human body, with these three materials are reported on the Figure 11d and e, respectively. At 25°C, the initial rate of ASA is lower with materials I and II compared with PNIPAM (Figure 11d). The releasing curves of BC materials


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overlap during the first 6 h. So, as expected, hydrogel enclosure in the bicontinuous structure limits the surface area in contact with the aqueous solution and thus slow down desorption of the active ingredient in the early hours of immersion. At 37°C, the most surprising feature is the initial rate of ASA releasing (Figure 11e). ASA releasing from BC material I may be slowed down by the tortuosity of the bicontinuous structure compared to PNIPAM single network but the rate turns out to be even lower with material II. So, it fit the scenario ruled out from the surface analyses of the dehydrated BC samples (Figure 8). Hydrogel is hydrated at 25°C with an opened structure from which ASA can still diffuse as depicted in the Figure 8 for the hydrated BC sample. In material I PtBMA does not change its shape when the hydrogel is collapsing at 37°C whereas in material II PBA extends when the hydrogel collapse. So, the surface exchange should be smaller in material II and the ASA releasing rate is thus reduced. The analyses carried out on the bicontinuous materials directly provide information on the reason why material II, composed of a poly (butyl acrylate) network is the one that releases the slowest the aspirin. The higher PNIPAM confinement reduces the exchange surface and thus slows down the drug release. The rubber-like hydrophobic polymer plays the role of in situ created transport barriers.44

4. CONCLUSION In this study, we have shown that it was possible to synthesize bicontinuous materials with continuous hydrophilic domains of PNIPAM network entangled with continuous hydrophobic polymer domains. During the materials synthesis, the dual phase continuity is imposed by the bicontinuous microemulsions used as template. A particular attention was paid to the microemulsion formulations using a surfmer to preserve the one-to-one replication of the


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bicontinuous nanostructure after polymerization. Two bicontinuous samples were synthesized. Their essential difference is the composition of their hydrophobic component: first material is based on stiff poly(tert-butyl methacrylate) and second rubber-like material on poly(butyl acrylate). The surface properties were studied to give the extent of the influence of the hydrophobic partner on the materials properties. Upon drying, hydrophobic parts are exposed at the surface and make the sample more hydrophobic than the single PNIPAM network. Once the sample is fully hydrated the water swelling of the hydrophilic domains rubs out the surface roughness. Materials bulk properties were obviously examined given the well-known absorption/release properties of PNIPAM. In the same way as PNIPAM, these materials have reduced hydrophilic behaviors when the temperature increases. The preservation of the volume phase transition for these materials allows considering them as drug carrier for thermal controlled release. PNIPAM, which will play the role of the active ingredient reservoir, is confined in the bicontinuous structure. Hence, we capitalized on the inherent tortuosity of bicontinuous microemulsions to conceive drug delivery device. Such a structure slows down the releasing without hindering it, and thus especially avoids the burst effect that can be observed when delivering a medicine such as aspirin, used as model drug. At first glance the particularly slow drug release from the poly(butyl acrylate) based sample may seem counterintuitive but it fit the scenario ruled out from the surface analyses of the dehydrated BC samples. In BC material the low Tg hydrophobic polymer extends when the hydrogel collapse, which induce reduction of the exchange surface with water and so of the drug releasing rate. The soft hydrophobic polymer plays the role of in situ created transport barriers. Nevertheless, all the loaded aspirin in the two bicontinuous structure remains still available since most of it ends up being desorbed.


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Beyond the targeted application described in this paper, soft template based on bicontinuous micromemulsion offers huge potential. Among other applications the development of proton conducting polymer membranes for PEMFC, polyelectrolyte membranes for batteries or nanoporous monoliths for filtration or chromatography could be envision for instance. ASSOCIATED CONTENT Supporting Information is available free of charge on the ACS Publications website. It contains Properties of the surfmer Brij 35Surf, Effect of the initiation locus for the radical polymerization and temperature effect on drug release.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] ORCID: Cedric Vancaeyzeele: orcid.org/0000-0002-9748-1562 Present Addresses † ICMN - UMR 7374- CNRS - Université d’Orléans, 1b rue de la Férollerie 45071 Orléans cedex 2

ACKNOWLEDGMENT The authors would like to thank the project SESAME (Comicer project) supported by “Ile de France” region that has allowed the acquisition of confocal laser scanning microscope.


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Table of Contents:


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Nanostructured Thermal Responsive Materials Synthesized by Soft

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