Self-Assembly of Oligosaccharide-b-PMMA Block Copolymer Systems

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Self-assembly of oligosaccharide-b-PMMA block copolymer systems: Glyco-Nanoparticles and their degradation under UV exposure Karine Zepon, Issei Otsuka, Cécile Bouilhac, Edvani Curti Muniz, Valdir Soldi, and Redouane Borsali Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b00212 • Publication Date (Web): 07 Apr 2016 Downloaded from http://pubs.acs.org on April 16, 2016

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Self-assembly of oligosaccharide-b-PMMA block copolymer systems: Glyco-Nanoparticles and their degradation under UV exposure Karine M. Zepon,1,2,3 Issei Otsuka,1, 2 Cécile Bouilhac,4 Edvani C. Muniz5, Valdir Soldi,3 Redouane Borsali1,2 * 1

University Grenoble Alpes, CERMAV, F-38000 Grenoble, France 2

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CNRS, CERMAV, F-38000 Grenoble, France Cedex 9, France.

Departamento de Química, Universidade Federal de Santa Catarina, CEP - 88040-900 Florianópolis − Santa Catarina, Brazil.

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Institut Charles Gerhardt Montpellier UMR5253 CNRS-UM-ENSCM, Equipe Ingénierie et

Architectures Macromoléculaires, Université Montpellier – Bâtiment 17 – cc1702, Place Eugène Bataillon, 34095 Montpellier cedex 5, France 5

Grupo de Materiais Poliméricos e Compósitos, GMPC—Departamento de Química, Universidade Estadual de Maringá, CEP 87020-900, Maringá, Paraná, Brazil.

Corresponding author: R. Borsali; e-mail: [email protected]

ACS Paragon Plus Environment

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Abstract

This paper discusses the self-assembly of oligosaccharide-containing block copolymer and the use of ultraviolet (UV) to obtain nano-porous glyco-nanoparticles by photo-degradation of the synthetic polymer block. Those glyco-nanoparticles consisting of oligosaccharide-based shell and a photo-degradable core domain were obtained from the self-assembly of maltoheptaoseblock-poly(methyl methacrylate) (MH-b-PMMA48) using the nanoprecipitation protocol. MH-bPMMA48 self-assembled into well-defined spherical micelles (major compound) with a hydrodynamic radius (Rh) of ca. 10 nm and also into large compound micellar aggregates (minor compound) with an Rh of ca. 65 nm. The oligosaccharide shells of these glyco-nanoparticles were cross-linked through the Michael-type addition of divinyl sulfone under dilute conditions to minimize the inter-micellar cross-linking. The core domain photo-degradation of the cross-linked glyco-nanoparticles was induced under exposure to 254 nm UV radiation, resulting in porous glyco-nanoparticles with an Rh of ca. 44 nm. The morphology of the cross-linked shell and the core photo-degradation of these glyco-nanoparticles were characterized using static light scattering (SLS), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance (1H NMR), field emission gun-scanning electron microscopy (FEG-SEM), and transmission electron microscopy (TEM). The innovative aspect of this approach concerns the fact that after removing the PMMA domains, the porous nanoparticles are mostly composed of biocompatible and non-toxic oligosaccharides.

Keywords. Block Copolymer, Self-Assembly, Nanoparticles, Micelles, UV degradation.


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TOC

Introduction The ability of block copolymers (BCPs) to self-assemble in a selective solvent has been shown to be a powerful tool to create nanostructures with different morphologies, such as, spherical micelles, cylindrical micelles, vesicles and bilayers,1–3 whose applications include drug delivery,4,5 nanoreactors,6–8 and nanotemplates.9–13 The latter targeted/potential application has gained the attention of researchers due to the possibility of preparing core-shell type structures, i.e., a core consisting of a solvophobic block and a shell consisting of solvophilic block,14 which allow the cross-linking reactions to be performed in the core domain15,16 or throughout the shell domain.10,11,17 The shell cross-linking reaction provides the structural stabilization required to produce hollow nanoparticles from solid nanoparticles18,19 by removing the core domain using physical and chemical procedures.20


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Hollow nanoparticles derived from micelles were reported for the first time in the pioneering work conducted by Wooley et al. in the late 1990s, where self-assembled poly(acrylic acid)block-polyisoprene micelles with cross-linked shells were used as templates to prepare hollow nanoparticles by removing of core domain via a chemical procedure.11 Since then, the potential application of nanostructured objects obtained through the self-assembly of a block copolymer as a structural template to produce hollow nanoparticles has been investigated21, due to the unique properties of these structures, such as large surface area,22 encapsulation of large quantities of guest molecules,20 low density, excellent permeation and penetration characteristics.23 Based on these remarkable properties, we evaluated the feasibility of producing hollow nanoparticles, using oligosaccharide-based block copolymer and their self-assemblies leading to glyconanoparticles, as the template. In this study, we firstly prepared glyco-nanoparticles in aqueous media consisting of crosslinkable maltoheptaose (MH) as the shell and photo-degradable poly(methyl methacrylate) (PMMA) as the core through the nanoprecipitation of an amphiphilic block copolymer, MH-bPMMA48, These glyco-nanoparticles were then reacted with divinyl sulfone (DVS) as a crosslinking agent for the MH block under dilute conditions. In this process, the shell domains of the glyco-nanoparticles (MH block) were covalently cross-linked via a Michael-type addition mechanism. Finally, the PMMA domains of these large compound micelles (LCMs) were removed by photo-degradation when exposed to UV radiation at a wavelength of 254 nm.24 To the best of our knowledge, this is the first example of porous glyco-nanoparticles obtained from the self-assembly of oligosaccharide-containing block copolymers.


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Experimental Materials MH-b-PMMA48 (subscript denotes the degree of polymerization of the corresponding block) was synthesized according to our previous report.25 Water was purified in a Milli-Q® water purification system (18.2 mΩ cm-1) (Purelab, ELGA). Acetone (99+% purity) was purchased from Carlos Erba Reagents and used as received. Divinyl sulfone (DVS, 98+% purity) was purchased from Sigma-Aldrich and used as received. Rotilabo® cellulose acetate (CA) filters (0.20 µm) were purchased from Carl Roth GmbH.

Block Copolymer Self-Assembly The block copolymer self-assembly was afforded applying the nanoprecipitation protocol.26 In a typical procedure, 3 mg of MH-b-PMMA48 was dissolved in 1 mL of a binary acetone/water solvent mixture (0.78/0.22 (w/w)) and the solution was allowed to stand under stirring overnight to achieve complete dissolution. The copolymer solution was then slowly added (0.5 mL h-1) to 3 mL of Milli-Q® water and stirred at 50 rpm for 3 h. Acetone was removed under reduced pressure at 30 °C and the self-assembled solution was ultimately filtered using the CA 0.2 µm filter.

Cross-linking of Glyco-nanoparticles’ Shell Briefly, 0.5 mL of the self-assembled MH-b-PMMA48 solution (1 g L-1) was added dropwise into a flask containing 10 µL of DVS (molar ratio of DVS:MH unit was 1:1) previously solubilized in 1.5 mL of Milli-Q® water and kept under continuous stirring (150 rpm) at room temperature. The


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cross-linking reaction was allowed to proceed for 24 h, and the solution was then filtered through the CA 0.2 µm filter.

Ultraviolet (UV) Radiation The cross-linked self-assembled MH-b-PMMA48 solution was poured into a 10-mm diameter cylindrical quartz tube and then exposed to UV radiation for different time intervals (wavelength of 254 nm with a laser energy density of 2.5 J cm-2). As the control, a non-cross-linked selfassembled MH-b-PMMA48 solution was prepared and submitted to UV exposure following the same protocol applied to the cross-linked sample.

Characterization of Glyco-nanoparticles Static Light Scattering (SLS) and Dynamic Light Scattering (DLS) SLS and DLS measurements were carried out using an ALV/CG6-8F goniometer equipped with a 35 mW red helium-neon linearly polarized laser (wavelength of 632.8 nm) and an ALV/LSE5004 multiple tau digital correlator with a 120 ns initial sampling time. In the SLS mode, the scattered light intensity was acquired at a scattering angle of 90° with a counting time of 120 s. MH-b-PMMA48 solutions in the mixture of acetone and water were loaded in 10-mm diameter cylindrical cells and then immersed in a thermostated toluene bath at 25 ± 0.5 °C. In the DLS mode, cylindrical quartz tubes containing the cross-linked or non-cross-linked self-assembled MH-b-PMMA48 dispersions were immersed in a thermostated toluene bath at 25 ± 0.5 °C. The data were acquired with the ALV correlator control software, and the counting time for the sample was typically 300 s at each different scattering angle ranging from 40 to 140° (i.e., 9.04 10-3 ≤ q = (4πn/λ )sin(θ/2) nm-1 ≤ 2.48 10-2 in pure water where q corresponds to the scattering


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vector modulus, n represents the refractive index of the pure solvent (1.332 for water at 25 °C), θ is the scattering angle (i.e., the observation angle) and λ designates the wavelength of the incident light) with a step of 10°. The relaxation time distribution (τ) was obtained using CONTIN analysis of the autocorrelation function (g(2) -1).27 From the apparent diffusion coefficient (D), obtained by plotting the relaxation frequency, Γ (τ-1) vs. the square of scattering vector modulus (q2), the Rh value could be determined using the Stokes–Einstein relation 1:

R =

(1)

where κ is the Boltzmann constant, Τ is the temperature, D is the apparent diffusion coefficient and η is the medium viscosity. The radius of gyration (Rg) was determined from the elastic part (I(q)) of the scattered intensity using the Guinier approximation following relation 2:

= −

(2)

where I is the scattering intensity and I0 is the scattering intensity at q = 0.

Fourier Transform Infrared Spectroscopy (FTIR) The cross-linking of the shell of the glyco-nanoparticles was evaluated by FTIR analysis. IR spectra were recorded using a Perkin-Elmer Spectrum RXI FTIR spectrometer in the range of 4000 to 400 cm-1. Firstly, in the self-assembled MH-b-PMMA48 solution obtained applying the nanoprecipitation protocol, the shells of the glyco-nanoparticles were cross-linked with DVS. The excess of DVS was then removed from the cross-linked self-assembled dispersion through dialysis against a very large volume of deionized water in 2 kDa-cutoff membranes tubing over 48 h. During this time, the water was exchanged 4 times at regular intervals. The resulting sample was then lyophilized for 24 hours. Subsequently, 3 mg of each sample, i.e., the lyophilized cross-linked self-assembled MH-b-PMMA48 dispersion, pure MH-b-PMMA48


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copolymer and pure DVS, were separately milled and mixed with 100 mg of potassium bromide (KBr) and pressed into pellets for the IR measurements.

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H Nuclear Magnetic Resonance (1H NMR)

Briefly, 3 mg of MH-b-PMMA48 was firstly dissolved in 1 mL of a binary acetone-d6/deuterated water solvent mixture (0.78/0.22 (w/w)), and then slowly added (0.5 mL h-1) to 3 mL of deuterated water under constant stirring (50 rpm). The acetone was removed under reduced pressure at 30 °C. 1H NMR measurements were carried out on a Bruker Avance DRX-400 spectrometer (400 MHz) aiming to assess the presence of by-products arising from the PMMA photo-degradation. The sample was exposed to UV radiation for different time intervals (0, 4 and 24 h). 1H NMR data were processed using an ACD/NMR processor.

Transmission Electron Microscopy (TEM) TEM was carried out using a CM200 Philips microscope (Hillsboro, OR, USA) operating at 80 kV and the images were recorded on Kodak SO163 films. The cross-linked self-assembled MHb-PMMA48 dispersion, with and without exposure to UV radiation, were dropped onto a glowdischarged carbon-coated copper grid and negatively stained with 2 % (w/v) uranyl acetate.

Field Emission Gun-Scanning Electron Microscopy (FEG-SEM) FEG-SEM was performed using a FEI/QUANTA FEG 250 microscope (Hillsboro, OR, USA) operating at an accelerating voltage of 8 kV. The cross-linked self-assembled MH-b-PMMA48 dispersion, with and without exposure to UV radiation, were dropped onto silicon substrates, dried in air and then coated with a 1 nm-thick layer of platinum/carbon.


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Results and discussion Solutions of MH-b-PMMA48 glyco-nanoparticles were prepared using nanoprecipitation technique. In this method, the first step is to select a solvent which is thermodynamicallyappropriate for both blocks, in order to dissolve the copolymer molecules as single chains.28 Accordingly, solubility tests were carried out considering our previous report on the selfassembly of MH-b-PMMA48 in water and acetone.25 Initially, MH-b-PMMA48 samples were dissolved in mixture solvents of acetone and water having various weight fractions and their normalized scattered light intensities (ISCN =

) were measured using the SLS setup (Fig. 1).

Figure 1. Normalized scattered light intensity (ISCN) of MH-b-PMMA48 solutions (1 g L-1) in mixture solvents of acetone and water (measured at a scattering angle of 90° at 25 °C) vs. weight percentage of water in the mixture. Graph inset shows the solution behaviour for the more concentrated solutions of MH-b-PMMA48 (5 g L-1).


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As illustrated on Figure 1, the ISCN value varied as a function of the weight fractions of acetone and water. The increasing of water contents in the medium change differently the solubility of each block, which increases for MH (hydrophilic block) and decreases for PMMA (hydrophobic block). Such behaviour leads to a decrease in the ISCN values with a minimum value at a binary acetone and water solvent mixture of 0.78/0.22 (% w/w). Considering that the scattered light intensity is proportional to the product of the copolymer concentration and aggregate molar mass29 in dilute regime, the smallest ISCN value should be associated to the dissociation of the of MH-b-PMMA48 aggregates into molecularly dissolved chains (single chain). Interestingly, the weight fraction of the PMMA block in the BCP (fPMMA = 0.81) is similar to the weight fraction of acetone (facetone = 0.78) in the solvent mixture (acetone/water = 0.78/0.22 (% w/w)) where MH-bPMMA48 exists as single chains. A similar result was obtained in our previous study for the diblock copolymer composed of maltoheptaose and polystyrene.30

The size distribution of MH-b-PMMA48 dissolved in a binary acetone and water solvent mixture (acetone/water = 0.78/0.22 (% w/w) was investigated by DLS (data not shown). The Rh value was determined as ca. 4.1 nm from the slope of the Γ vs. q2 plot using the Stokes-Einstein relation. This size was consistent with theoretical value of ca. 3.6 nm, calculated based on the average unperturbed end-to-end distance of a polymer chain (〈!〉 = . √%, where is the length of one repeat unit ( PMMA ~0.3 nm and Maltose ~0.5 nm) and N is the number of repeating units).31 The similarity between the experimental and theoretical values confirms the dissolution of the MH-b-PMMA48 as single chains in the binary acetone/water (0.78/0.22 % w/w) mixture.


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Subsequently, the slow addition of the well-dispersed (single chain) MH-b-PMMA48 solution into water was demonstrated to induce the copolymer self-assembly according to the nanoprecipitation protocol. The self-assembled MH-b-PMMA48 solution was then analyzed by DLS. Figure 2 (A-B) shows the autocorrelation function (g(2) -1) and the hydrodynamic radius distribution (at a scattering angle of 90° at 25 °C) with the respective dependence of the relaxation frequency Γ (1/τ) on the square of the wave vector (q2).

Figure 2. (A) Autocorrelation function (g(2) -1) of the self-assembled MH-b-PMMA48 solution measured at a scattering angle of 90° at 25 °C and (B) the respective dependence of the relaxation frequency (1/τ) on the square of the wave vector (q2). Graph inset shows the distribution of the hydrodynamic radius (A (q,t)) (z-averaged hydrodynamic distributions) at a scattering angle of 90°.

The autocorrelation function (g(2) -1) shows two exponential decays with two diffusive modes. The fast diffusive mode corresponds to the micelles whereas the slower mode can be attributed to compound micellar aggregates, so-called LCMs. These LCMs are assumed to be clusters formed by micelles that aggregate each to other through nonionic maltoheptaose shells.32


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Furthermore, the Brownian motion of the self-assembled glyco-nanoparticles was confirmed by the linear q2 dependence of the relaxation frequency for both diffusive modes.33 The Rh values were calculated as ca. 10 nm (micelles) and ca. 65 nm (LCMs) through the Stokes-Einstein relation using the D value obtained from the slope of Γ vs. q2 plot (D fast mode = 2.37 10-11 m2 s1

; D slow mode = 3.84 10-12 m2 s-1). It should be noted that the highest peak intensity related to

the LCMs does not mean there are a greater number of the LCMs in solution. In fact, the micelles in the self-assembled MH-b-PMMA48 solution represented more than 98% of the total nanoparticles. The minor percentage of LCMs could be confirmed from DLS and TEM analysis. The results in a number-weight distribution (Fig. 3A) shows that the MH-b-PMMA48 selfassemble mainly into micelles with a radius of ca. 16 nm and well-distributed spherical shape (Fig. 3B). It is assumed that these spherical micelles consist of a hydrophobic core composed of PMMA surrounded by hydrophilic maltoheptaose as the shell.

Figure 3. (A) DLS, number-weight distribution and (B) TEM image of the glyco-nanoparticles obtained by self-assembly of MH-b-PMMA48 in water. Insert is an enlarged view of the indicated area for the non-cross-linked glyco-nanoparticles.


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The shells of the glyco-nanoparticles composed of the MH block were cross-linked using DVS in order to obtain a porous structure after the PMMA core photo-degradation. To minimize the cross-linking of the inter-micellar shell, the self-assembled solution was diluted 3-fold in MilliQ® water containing DVS (DVS:MH unit molar ratio of 1:1) and stirred for 24 h. The occurrence of cross-linking was confirmed by FTIR spectroscopy (Fig. 4). The hydroxyl groups in MH were cross-linked via Michael-type addition using DVS as the cross-linker. The peaks observed on the FTIR spectrum related to the pure DVS (Fig. 4A-I) were assigned as follows:34 a strong peak at 1312 cm-1 associated with the asymmetric stretching vibration of ν[S=O] and a peak at 1130 cm-1 related to the symmetric stretching vibration of ν[S=O]. The peaks at 1610 cm-1 and 3010-3100 cm-1 were attributed to ν[C=C] and ν[=CH], respectively. The FTIR spectrum for the non-crosslinked self-assembled MH-b-PMMA48 solution (Fig. 4A-II) shows peaks at 3440 cm-1, 2952 cm1

, 1150 cm-1, and 1030 cm-1 which are associated with the symmetric and antisymmetric

stretching of ν[OH], ν[CH2] and ν[C-C] and the bending vibration of ν[C-OH], respectively.35 The FTIR spectrum for the self-assembled MH-b-PMMA48 solution cross-linked with DVS (Fig. 4A-III) shows a strong peak at 1312 cm-1 which correspond to the asymmetric stretching vibrations of ν[S=O] and also the peak at 1130 cm-1 associated to the symmetric stretching vibration of ν[S=O] (Fig. 4B). Moreover, the absence of the peak at 1610 cm-1 assigned as the ν[C=C] vibration and the shift of the ν[=CH] stretching to a lower wavenumber indicate that the DVS double bond (C=C) was opened during the cross-linking reaction forming the C-O-C bond. This result confirms the successful cross-linking of MH blocks by DVS.


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Figure 4. (A) FTIR spectra obtained from (I) pure DVS, (II) pure MH-b-PMMA48, and (III) cross-linked self-assembled MH-b-PMMA48 glyco-nanoparticles.

The effect of UV radiation time on the morphological properties of the cross-linked and noncross-linked self-assembled MH-b-PMMA48 glyco-nanoparticles was investigated by DLS and the results are shown in Figure 5 (A-B). Photo-degradation of the PMMA block was accomplished by exposure to UV radiation (approximately 2.5 J cm-2) of the cross-linked and non-cross-linked self-assembled glyco-nanoparticles. As shown in Figure 5A, cross-linked and non-cross-linked self-assembled MH-b-PMMA48 glyco-nanoparticles show apparent Rh values of ca. 65 nm and 70 nm, respectively, prior to the UV exposure. After 2 h of UV exposure, the cross-linked LCMs showed a remarkable decrease in the apparent Rh value from ca. 65 to 40 nm, this value remaining almost constant over 48 h. Visually, after 48 h of UV radiation, the crosslinked LCMs showed a bluish aspect, which is normally associated with the Tyndall effect36 due


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to the presence of large nanoparticles37 (Fig. 5B). On the other hand, after 2 h of UV exposure, the non-cross-linked LCMs showed two different apparent Rh values, the first with a value of ca. 19 nm and the second with a value of ca. 74 nm. The Rh values changed over time, revealing the presence of large aggregates (visible to the naked eye) after 48 h.

Figure 5. (A) Apparent Rh values for () non-cross-linked (diffusive slow mode), () noncross-linked (diffusive fast mode) and () cross-linked self-assembled glyco-nanoparticles as a function of UV exposure time measured at a scattering angle of 90° at 25°C and (B) the appearance of the self-assembled non-cross-linked (left-side) and cross-linked (right-side) MHb-PMMA48 solutions after 48 h of exposure to UV radiation. Regarding the non-cross-linked LCMs, we hypothesize that the large aggregates seen after UV exposure could be clusters formed by the maltoheptaose block containing small water-insoluble fragments of PMMA or by water-insoluble oligo-MMA formed through the photo-induced repolymerization of the photodegraded MMA. As the non-cross-linked LCMs do not have structural support to prevent their disassembly after core photo-degradation, it is reasonable to


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expect that the LCMs partly disassembled and then re-aggregated before the PMMA blocks were totally photo-degraded. The morphology and photo-degradation process of the cross-linked glyco-nanoparticles were also studied by the 1H NMR and are illustrated in Figure 6. The absence of the signal assignable to –OCH3 groups in PMMA (δ ~3.7 ppm) in the spectrum related to the self-assembled MH-bPMMA48 solutions prior to the UV exposure (time zero) indicates that the PMMA segments make up the core domain, which leads to a reduction in the proton mobility.38 On the other hand, the signal corresponding to maltoheptaose (H1, α 1-4 linkages)39 could be clearly observed at δ ~5.1 ppm, indicating that the oligosaccharide shell segments are completely solvated by water. After 4 h of UV exposure, the signal assignable to –OCH3 groups in PMMA (δ ~3.7 ppm) and the signal assignable to -C=CH2 groups derived from unsaturated end-groups in the PMMA oligomers40 (δ ~5.6 ppm) were clearly observed. The presence of the signal related to the C=CH2 is consistent with the PMMA photolysis mechanism previously proposed by Wochnowski et al.,41 confirming that the PMMA photolysis was successfully achieved through exposure to UV radiation. It should be noted that the broaden signals due to PMMA moiety after 24 h UV exposure indicates re-polymerization of the photo-degraded MMA, which could also contribute to the formation of the aggregates confirmed by DLS analysis described above.


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Figure 6. 1H NMR spectra of self-assembled MH-b-PMMA48 glyco-nanoparticles in D2O when exposed to UV radiation for different time intervals.

In order to gain a better understanding regarding the impact of UV radiation on the morphological and dimensional properties of the cross-linked LCMs, DLS and SLS measurements were carried out. In the DLS results in Figure 7 (A-B), the autocorrelation function (g(2) -1) shows a single exponential decay with only one diffusive mode. This diffusive mode (seen for all scattering angles) probably corresponds to the photo-degraded LCM, which shows the q2 dependence of the relaxation frequency, confirming the Brownian motion.33 The D value was determined from the slope of the Γ vs. q2 plot and applying the Stokes-Einstein relation the Rh was the calculated as ca. 44 nm.


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Figure 7. (A) Autocorrelation function (g(2) -1) of cross-linked self-assembled MH-b-PMMA48 solution after 24 h of exposure to UV radiation measured at a scattering angle of 90° at 25 °C and (B) the respective dependence of the relaxation frequency (1/τ) on the square of the wave vector (q2). Graph inset shows the distribution of the hydrodynamic radius (A (q,t)) (z-averaged hydrodynamic distributions) at a scattering angle of 90°.

The effect of UV radiation on the cross-linked LCM morphology was investigated by SLS using the ρ-parameter value (ρ-value). The ρ-value =

&' &(

provides important information regarding the

topology of the nanoparticle. It is well-known that for a homogeneous sphere the ρ-value is 0.775, whereas for a hollow sphere the ρ-value is 1.42 Therefore, the radius of gyration (Rg) of the cross-linked LCMs after exposure to UV radiation was determined using the Guinier approximation (Fig. 8), assuming that at very small angles (RGq < 1.3) the intensity could be well represented by Eq. (2).


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Figure 8. Guinier plot calculated from the elastic light scattering intensity of the cross-linked self-assembled MH-b-PMMA48 solution after 24 h of UV exposure.

The Rg value determined from the slope of the ln I vs. q2 plot using the Guinier approximation was ca. 38 nm, which leads to a ρ-value of 0.86. This ρ-value was smaller than that expected for a hollow sphere. Although unexpected, this ρ-value has been obtained by other researchers17,43 in studies related to the preparation of hollow structures. Different hypotheses have been considered to explain this lower ρ-value, one being related to the possibility of the LCMs (also designed as multicompartmental micellar structures) being only partially hollow,43 and another to the large thickness of the hollow wall.17 Taking into account that the LCMs are formed from several individual micelles44 and in view of the aforementioned hypotheses, we propose that the porous LCMs are formed of simple hollow micelles after exposure to UV radiation. In order to


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further investigate the effect of UV radiation on the cross-linked MH-b-PMMA48 morphology, FEG-SEM analysis was carried out and the images obtained are shown in Figure 9 (A-B).

Figure 9. FEG-SEM images obtained from of cross-linked self-assembled MH-b-PMMA48 glyco-nanoparticles (A) before and (B) after exposure to UV radiation (scale bar, 200 nm).

The FEG-SEM image obtained prior to exposure to UV radiation (Fig. 9A) showed small spherical micelles and also well-defined spherical cross-linked LCMs having diameters between 25 and 100 nm. After exposure to UV radiation (Fig. 9B), the LCMs are partially collapsed and the presence of some cavities corroborates the DLS/SLS results towards to the UV exposure produces hollow structure. TEM analysis was also conducted in order to obtain further information on the effect of UV radiation on the cross-linked MH-b-PMMA48 morphology, as illustrated in Figure 10(A-B). Prior to exposure to UV radiation (Fig. 10A), spherical micelles and well-defined spherical cross-linked LCMs having diameters between 20 and 90 nm were observed in the self-assembled MH-b-PMMA48 solution. On the other hand, after UV radiation (Fig. 10B), LCMs with well-defined shape were observed; however, a more accurate characterization of the internal structure could not be obtained due to the low contrast between the PMMA and maltoheptaose, even with negative staining. Nevertheless, the TEM images,


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which look similar before and after UV-exposure, are consistent with the SEM images in terms of diameter range but the FEG-SEM experiments gave more details about the porosity of the glyco-nanoparticle’s surface as illustrated in Figures 9.

Figure 10. TEM images obtained for cross-linked self-assembled MH-b-PMMA48 glyconanoparticles (A) before and (A) after exposure to UV radiation (scale bar, 200 nm). Insert is an enlarged view of the indicated area for the crosslinked glyco-nanoparticles before (upper left) and after (upper right) UV exposure.

Conclusions A MH-b-PMMA48 solution was prepared by dissolving the BCPs in a binary acetone and water solvent mixture of 0.78/0.22 (% w/w), where the MH-b-PMMA48 molecules behaved as single chains. The BCPs self-assembled into well-defined glyco-nanoparticles with hydrodynamic radius (Rh) values ranging from ca. 10 nm to ca. 65 nm by nanoprecipitation protocol. The shells of the glyco-nanoparticles were successfully cross-linked by DVS, as indicated by the FTIR


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results. In addition, the glyco-nanoparticle core was degraded on exposure to UV radiation and porous glyco-nanoparticles were obtained, as evidenced by the TEM and FEG-SEM images. The promising applications of these porous glyco-nanoparticles for delivery vehicles are supported by the fact that the degradation of PMMA moiety could facilitate the release of encapsulated hydrophobic guest compounds and the remaining vehicles are mostly composed of biocompatible and non-toxic oligosaccharides. Acknowledgment This work was sponsored by CNPq (Proc. 400702/2012-6), CNRS, Institut Carnot PolyNat, Greenanofilms (Grant agreement #603519 ) and Labex Arcane (ANR-11-LABX-0003-01). The authors are very grateful to Christine Lancelon-Pin for TEM and FEG-SEM and Christophe Travelet for light scattering data. References (1)

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Self-Assembly of Oligosaccharide-b-PMMA Block Copolymer Systems

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