Marianne Gaborieau

Hank De Bruyn

Siyabonga Mange

Patrice Castignolles

Andreas Brockmeyer

Robert G Gilbert
email: b.gilbert@uq.edu.au

Synthesis and Characterization of Synthetic Polymer Colloids Colloidally Stabilized by Cationized Starch Oligomers

Keywords: colloids, polysaccharides, cerium, graft copolymers, nuclear magnetic resonance (NMR), methyl methacrylate

A method is developed for anchoring cationized starch oligomers as electrosteric stabilizers onto synthetic latexes, using Ce 4+ to create free-radical grafting sites on the starch. Using -amylase to randomly cleave starch, and N-isopropyl acrylamide, whose corresponding polymer exhibits a lower critical solution temperature, thermosensitive block oligomers of oligosaccharide and synthetic polymer are synthesized. Above 30 °C, they form self-emulsifying nanoparticles ("frozen micelles") from which a synthetic latex is grown after addition of methyl methacrylate, the collapsed NIPAM-containing entities functioning as in-situ seed. The grafting efficiency is high (as shown by a new 13 C solid-state NMR method), and the resulting latex is very colloidally stable.

INTRODUCTION

Polymer colloids that are stabilized by grafted oligosaccharides have many potential applications, e.g. in paper coating, [1][2][3] soil protection against water erosion, 4 drug delivery 5,6 and as models to study the biological activity and colloidal stability of living cells. 5,7,8 Oxidation of the oligosaccharides results in a radical on the chain, which will then initiate the polymerization of a synthetic monomer and finally yield a copolymer, thereby resulting in grafting. Several reagents have been used in the past for such an oxidation of oligosaccharides: potassium persulfate, 7 ozone 2 and Cerium(IV) in an acidic medium. 2,6,8,9 Latexes sterically stabilized by grafted linear starch oligomers (which are uncharged) have moderate colloidal stability. 8 The use of a cationized starch rather than a native neutral one is expected to lead to species with enhanced colloidal stability, by combination of the cationic charge and steric-stabilizing effects of the grafted starch, resulting in an electrosteric stabilizer. [START_REF] Bassett | Polymer Colloids II[END_REF][START_REF] Napper | Polymeric stabilization of colloidal dispersions[END_REF] Latexes stabilized with fragments of cationized starch have previously been synthesized by Gugliemelli et al. After gelatinizing the cationized starch to disperse it (i.e., heating in water to page 3 break up the crystalline fractions of the starch), they grafted synthetic polymers onto the starch using cerium(IV) as an initiator for the polymerization of acrylonitrile 9,[START_REF] Gugliemelli | [END_REF]13 or chloroprene; 14 the mixture was finally sonicated to obtain a latex. The grafting step presumably led to a structure composed of a branched polysaccharide scaffold decorated with synthetic polymer particles. 2 Subsequent sonication greatly reduced the viscosity of the mixture, possibly due to cleavage of starch chains. 2,9 The systems so produced would have been ill-defined: the length of the polysaccharide fragments produced by sonication, which would affect the stability of the final latex, was not controlled and not reported. Well defined latexes stabilized by oligosaccharides may be obtained by grafting the initiator of the polymerization directly on the polysaccharide, but this synthetic pathway is a tedious multi-step one. [15][16][17] More accessible, well-defined latexes stabilized by fragments of another cationic polysaccharide, chitosan, have been synthesized and tested for medical applications. 5,18,19 The aim of the present paper is to present and compare syntheses and characterization of accessible, well-defined latexes stabilized by grafted linear or branched fragments of cationized (1→4)--glucans, obtained from the degradation of cationized starch.

The cationized starch used in the present work is Amylofax, a commercial modified starch bearing quaternary ammonium groups and obtained by functionalization of native potato starch using a wet process, which destroys the granular structure and thus ensures a homogeneous functionalization of the chains. 20 It was also chosen because it is more soluble in water than native starch, 20,21 in particular at high degrees of substitution, 22 and thus is expected to be a better stabilizer than the neutral starch used in earlier work. 8 To fulfill the desired goal, Amylofax needs to be degraded, as the Amylofax molecules are bigger than the polymer colloid it should stabilize, and (unless the size ratio were reversed) one might expect a 'Christmas tree' architecture to result: small domains of hydrophobic polymer decorating an amylopectin 'tree'. 3 The requisite degradation is implemented here by debranching or random cleavage. Cerium(IV) is chosen here to initiate the oxidation of starch and the subsequent polymerization of the synthetic monomer, following previous work. 3,8 Cerium(IV) has been used page 4 industrially to graft synthetic polymer onto starch and cellulose. 23,[START_REF] Hebeish | The chemistry and technology of cellulosic copolymers[END_REF] Initiation by potassium persulfate (commonly used in emulsion polymerization) was not used here, as it can initiate the synthetic monomer directly. Initiation by ozone was also not chosen, as it greatly degrades starch resulting in an excess of small fragments which can have deleterious effects on the final properties. 2 Cerium(IV) does not initiate directly on vinyl monomers, [START_REF] Moad | The Chemistry of Free Radical Polymerization[END_REF] and is known to complex and react rapidly with diols on neighboring carbons causing cleavage and, it is thought, production of a radical at the cleavage site. [START_REF] Moad | The Chemistry of Free Radical Polymerization[END_REF][START_REF] Mcdowall | [END_REF] Radicals borne by the oligosaccharides then initiate free-radical polymerization of synthetic monomers, yielding block oligomers or graft oligomers.

Several possible synthetic pathways will first be designed and described. Characterization methods for the oligosaccharides and the final latexes will then be detailed or developed. Latexes obtained by all synthetic pathways will finally be described.

EXPERIMENTAL SECTION Materials

Amylofax HS starch from AVEBE Deutschland GmbH is a potato starch substituted with hydroxypropyl-trimethyl-ammonium groups, following a wet process. 20 Isoamylase (Megazyme, 250 UmL -1 ), sodium acetate (Univar), ethanol (95 %, Aldrich), sodium hydroxide (Aldrich), D2O (99.9 %, Cambridge Isotope Laboratories Inc.), cerium(IV) ammonium nitrate (Ce(NH4)2(NO3)6, abbreviated CAN, 99.99 % metals basis, Aldrich), nitric acid (70 %, Riedel-deHaen), -amylase (Termamyl 120L from Novozyme), trisodium citrate (99%, Aldrich), calcium acetate hydrate (99.9%, Ajax Finechem), glacial acetic acid (analytical-reagent grade, Ajax Finechem) and lauroyl peroxide (97 %, Aldrich) were used as received. High-purity deionized water (Milli-Q) was used for all processes.

Methyl methacrylate (MMA, Mitsubishi Rayon, stabilized with 4-methoxyphenol) was passed through a column of basic alumina and distilled under reduced pressure (first and last 10 % page 5 discarded) to remove the inhibitor. The purified monomer was stored at 0 °C for no longer than 2 weeks before use. N-Isopropyl acrylamide (NIPAM, 97 %, Aldrich) was purified by recrystallization from hexane/toluene 65/35 v/v and dried at room temperature in a vacuum dessicator. N,N'-methylenebisacrylamide (BIS, 99 %, Aldrich) was used as received.

Low molecular weight poly(methyl methacrylate) (PMMA; Aldrich, 182230) and poly(Nisopropyl acrylamide) (PNIPAM, ca. 50 000 g•mol -1 ) homopolymers were used for the model blend.

Synthesis and characterization methods

Debranching of Amylofax

Amylofax HS was debranched following the protocol commonly used for native starch, 27 with slight modifications. 8 Briefly, 50 mg Amylofax were incubated at 50 °C for 18 h with 50 L isoamylase. Those quantities, used for investigating completeness of debranching by NMR, were scaled up for the grafting experiment.

Synthesis of electrostatically stabilized cationic seed latex

This synthesis, based on a recipe by van Berkel, 28 was detailed in a previous paper. 8 The critical micellar concentration (CMC) of the PMMA seed latex with dodecyltrimethylammonium bromide (DTAB) surfactant was determined previously. 8 The z-average diameter of the particles was 90 nm (measured by a back-scattering device, the Malvern high-performance particle sizer, HPPS), and the solids content was 10.9 wt % (measured by gravimetry).

Grafting of debranched Amylofax on the seed latex

The same protocol previously used for debranched neutral starch 8 was followed here with debranched Amylofax. 6.45 g of the PMMA seed latex were diluted with 3.2 g deionized water and stabilized further with 0.04 g of DTAB. The particles were swollen with 0.77 g of MMA at page 6 room temperature over 24 h, then brought to 40 °C. 500 mg of debranched Amylofax was added and the solution degassed for 20 min. A solution of 110 mg CAN in 4 mL of 0.2 molL -1 nitric acid was fed over 30 min with a syringe pump. The reaction was allowed to run overnight. The resulting latex was dialyzed against deionized water for 14 days with daily changes of the water.

Random cleavage of Amylofax with -amylase

16.95 g Amylofax HS were introduced in a flask, followed by 100 g deionized water, 0.13 g calcium acetate hydrate (25 % solution in water) and 0.63 g of a 1 % aqueous solution of Termamyl 120L, under stirring. The mixture was heated to 85 °C, then 2.22 g of a 1 % aqueous solution of Termamyl 120L solution were added. After 23 min, 31.66 g of glacial acetic acid were poured onto the reaction mixture to stop the cleavage reaction. The final total solid content was measured by gravimetry to be 11.6 wt %, which is also the oligosaccharide content.

First ab initio latex synthesis with cleaved Amylofax

4.92 g of the enzymatically randomly cleaved starch solution and 10 mL of a 0.2 mol.L -1 nitric acid solution were poured under stirring into a 100 mL round-bottom flask at 45 °C. The reaction mixture was purged with ultrapure nitrogen for 10 min. Then a 26.6 mL of a 0.08 mol.L -1 solution of CAN in 0.2 mol.L -1 nitric acid was added, immediately followed by 2 mL of MMA, and the medium then purged with nitrogen for a further 10 min. The reaction was allowed to run for a further 30 min, then the reaction mixture was cooled down to room temperature. 1.2 mL of 0.12 mol•L -1 trisodium citrate were added to 10 mL of reaction mixture, and the pH was adjusted to between 7 and 8 with 10 mol•L -1 NaOH. The addition of trisodium citrate prevents polymer precipitation during the addition of base. The latex was then dialyzed in deionized water to remove unreacted monomer, cerium ions and polysaccharide fragments, with water changes every 2 h until the color changed from greenish to opaque white. page 7

Synthesis of oligosaccharide-g-oligoNIPAM

In a 100 mL round-bottom flask at 29 °C, 5.5 g of the randomly cleaved starch solution and 10 mL of a 0.2 mol•L -1 nitric acid solution were poured under stirring. The reaction mixture was purged with nitrogen for 10 min. Then 26.6 mL of a solution of 1.17 g CAN in 0.2 mol•L -1 nitric acid was added, immediately followed by 1.87 g of NIPAM, and the medium was purged with nitrogen for a further 10 min. The reaction was allowed to run for a further 30 min.

Second ab initio latex synthesis with cleaved Amylofax, involving NIPAM

5.5 g of a 10 wt % enzymatically randomly cleaved starch solution was poured in a 100 mL round-bottom flask fitted with a septum, followed by 10 mL of 0.2 mol•L -1 nitric acid. The reaction mixture was purged with nitrogen at 29 ºC for 10 min before addition of 1.17 g of CAN dissolved in 26 mL of 0.2 mol•L -1 nitric acid. This was then followed immediately by addition of 1.5 g NIPAM dissolved in 10 mL deionized water and 0.11 g BIS dissolved in 10 mL deionized water. After 30 min the reaction temperature was raised to 60 ºC and allowed to equilibrate for 20 min before the addition of 0.5 g MMA containing 0.02 g of dissolved lauroyl peroxide. The reaction mixture was purged with nitrogen for 10 min and the reaction was allowed to run for a further 2 h. The final product was dialyzed in deionized water to remove cerium and any other water soluble entities.

H solution-state NMR of original and cleaved Amylofax

The debranched and randomly cleaved Amylofax samples were freeze-dried. Original, debranched and randomly cleaved samples were dissolved in D2O at 5, 20 and 50 mgmL -1 respectively (dynamic light scattering data suggest that Amylofax dissolves quickly and completely in water; see Figure S1 in the Supporting Information). Quantitative 1 H NMR spectra were then measured at 90 °C on a Bruker Avance spectrometer at a 1 H Larmor frequency of 300.13 MHz, with a 7.9 s 90° pulse, delays between scans of 10, 12.5 and 20 s, respectively, and 6360, 2400 and 1200 s transients, respectively. One-dimensional inversion-recovery page 8 experiments [START_REF] Claridge | High-resolution NMR techniques in organic chemistry[END_REF] were carried out to ensure that all signals of interest were quantitative under these conditions. The chemical shift scale was calibrated using the signal of HOD at 4.1 °C as an internal standard. [START_REF] Hoffman | [END_REF]

Transmission electron microscopy (TEM)

Dilute dispersions of polymer particles (about 0.05 wt %) were embedded on a carbon-coated copper grid and dried under vacuum. The grid containing the polymer particles was then placed in a Biofilter Philips CM120 transmission electron microscope equipped with a CCD camera and an accelerating voltage of 120 kV. The length scale was calibrated with a standard sample of polystyrene latex particles.

Isolation of grafted species by ultracentrifugation

A Beckman Coulter L-100 XP Ultracentrifuge with a 90 Ti rotor was used, for which 90,000 rpm generates a maximal relative centrifugal force of 694,000 g and an average relative centrifugal force of 503,000 g. The dialyzed latex was subjected to three successive ultracentrifugation runs at 90,000 rpm and 25 °C for 30 min. The centrifugate was redispersed in deionized water through sonication between runs. The final centrifugate, which should contain the oligosaccharide-g-PMMA or oligosaccharide-g-(PNIPAM-co-PMMA), was dried in an oven at 60 °C for 24 h.

Quantification of oligosaccharides grafted on latexes by solid-state NMR

The centrifugate was analyzed by solid-state 13 C NMR spectroscopy on a Bruker Avance 300 wide-bore spectrometer (Bruker Biospin Pty Ltd.), operating at 75 MHz for 13 C, using a 4 mm magic-angle spinning (MAS) probe (Bruker Biospin Pty Ltd.). The spectrometer was operated with XWIN-NMR software (Bruker Biospin GmbH). Samples were ground to a fine powder, packed in a 4 mm zirconia rotor with a KelF cap and spun at 4.5 kHz MAS at room temperature.

A 13 C single-pulse excitation (SPE-MAS) spectrum of the centrifugate was recorded with a 5.5 s 90° pulse and continuous wave decoupling (optimized on adamantane); 2640 transients were page 9

recorded with a delay of 20 s between scans, for a total experimental time of 14 h 45 min. A 13 C cross-polarization (CP-MAS) spectrum of the centrifugate was recorded with a 5.5 s 1 H 90° pulse, a 3 ms contact time and continuous wave decoupling (optimized on adamantane); 10240 transients were recorded with a delay of 3 s between scans, for a total experimental time of 9 h. 13 C CP-MAS spectra of pure Amylofax, pure PNIPAM and a model blend (containing exactly 62.33 mg Amylofax, 185.50 mg PNIPAM and 61.98 mg PMMA) were measured under the same conditions, with respectively 5060, 2848 and 22550 transients. The 13 C chemical shift scale was externally referenced to tetramethylsilane (TMS) at 0.0 ppm using adamantane by setting the CH resonance to 38.5 ppm. 31 Chemical shifts of PMMA are known from the literature. [START_REF]NMR spectra database of polymers[END_REF]

RESULTS AND DISCUSSION

Synthetic strategies

Grafting from the oligosaccharides is systematically done in this work using initiation by cerium(IV). Cerium(IV) complexes and reacts rapidly with diols on neighboring carbons causing cleavage and, most probably, production of a radical at the cleavage site. [START_REF] Moad | The Chemistry of Free Radical Polymerization[END_REF][START_REF] Mcdowall | [END_REF][START_REF] Pottenger | [END_REF] Studies of cellulose oxidation by cerium(IV) showed that it preferentially occurs at the 1,2-diol on the reducing chain end of the polysaccharide, and to a considerably lesser extent at 2,3-diols on the non-terminal anhydroglucose units. [START_REF] Mcdowall | [END_REF]34,35 Considering the similar chemical structures of starch and cellulose (both polymers of anhydroglucose units, respectively linked by (1→4)- and (1→4)- linkages), it is surmised that these observations are also valid for starch. Cerium(IV) in an acidic medium has been shown to cleave oligosaccharide chains; 36 however, the presence of a species reacting with the oligosaccharide radical limits this degradation. Both degradation methods used to obtain starch fragment in the present work are expected to yield short cationized starch fragments with one reducing end; in this case, the radicals are produced by cerium(IV) preferentially at the reducing end of these fragments. These radicals borne by the oligosaccharides initiate the free-radical polymerization of synthetic monomers, leading to the page 10 formation of block oligomers (or graft oligomers), when the radical is borne by the terminal (or non-terminal unit, respectively). This reaction mechanism is detailed in Scheme 1.

The first step in the strategy to synthesize latexes stabilized by cationized starch is to implement for this cationized starch a recently developed methodology for producing a latex stabilized by oligosaccharides: 8 debranching it, then anchoring it onto a seed latex to synthesize well-defined starch-graft-poly(methyl methacrylate) core-shell particles by second-stage emulsion polymerization (Scheme 2, top). The oligosaccharides used in the present paper are ionic, and thus very hydrophilic, and might behave differently from the neutral ones used previously during the emulsion polymerization. 8 Some alternative syntheses will also be explored, involving -amylase for starch degradation and an ab initio latex synthesis rather than a seeded one (Scheme 2, middle and bottom). In the ab initio pathways, the starch degradation can be carried out in the same reactor in which the polymerization is subsequently performed. To the best of our knowledge, those alternative synthetic pathways have not been used before with cationized starch oligomers.

In the second synthetic procedure (Scheme 2, middle), the oligosaccharides obtained by random cleavage of Amylofax by -amylase are branched and can be of various sizes (and thus bear various numbers of charges) depending on the particular cleavage conditions. Monomer and initiator will be added directly to the reaction mixture resulting from the cleavage experiment. Cerium(IV) will be used here to initiate the polymerization of a hydrophobic monomer, methyl methacrylate (MMA) by creation of a radical on the reducing end of the oligosaccharides. The resulting amphiphilic block-oligomers are expected to spontaneously form latex particles stabilized by branched oligosaccharides. The addition of monomer would then lead to formation of nanoparticles sterically stabilized by grafted starch chains. This ab initio procedure may suffer from a slow particle nucleation step through the self-assembly of the amphiphilic block copolymers. A different mechanism for the particle formation was thus also devised, involving a thermoresponsive synthetic intermediate.

page 11

This third synthetic pathway (Scheme 2, bottom) involves the synthetic monomer N-isopropyl acrylamide (NIPAM). The corresponding polymer, PNIPAM, is well known for exhibiting a lower critical solution temperature (LCST): 37,38 an increase in temperature decreases the polymer's water solubility due to increasing hydrophobic interactions. The LCST is the temperature at which the polymer solution undergoes a phase transition from a soluble state (i.e., random-coil form) to an insoluble state (i.e., collapsed or globular form) on elevating the temperature. The observed transitions with temperature are due to breakage and formation of hydrogen bonds between the PNIPAM amide groups and water, leading to deswelling and swelling, respectively. PNIPAM has been combined to other polymers to produce thermoresponsive core-shell latex particles with the PNIPAM shell covalently bound 39,40 or not 41,42 to the core, in order to obtain a thermoresponsive material. Our approach is different, as it consists in using the thermoresponsive property of PNIPAM during the synthesis. The rationale behind the use of NIPAM in the present work as a synthetic monomer to overcome the slow nucleation issue is the following: (i) at room temperature, NIPAM and PNIPAM are watersoluble, and thus should provide efficient grafting to water-soluble Amylofax fragments, (ii) above PNIPAM's LCST (32 °C), it is water-insoluble, and the grafted species will form selfemulsifying nanoparticles (sometimes called "frozen micelles", meaning micelles in which the surface-active components are supposed to be very slow to migrate from micelle to micelle) upon raising the temperature, (iii) above the LCST, polymerization can be continued inside the selfemulsifying nanoparticles: effectively an in-situ seeded emulsion polymerization. The thermoresponsive property of PNIPAM was similarly used as a method during synthesis to obtain well-defined latex particles by Pokhrel et al. 43 However, they used the PNIPAM collapsed domains only as a template for the particles, and the PNIPAM, not covalently linked to the latex particles, was subsequently extracted from the material. Our approach aims not only at obtaining defined latex particles, but also at anchoring water-soluble oligomers in the formed hydrophobic latex particles by a covalent link to a species embedded in it. These water-soluble oligomers subsequently play the role of colloidal stabilizers. Amylofax will be enzymatically cleaved using -amylase. Then cerium(IV) and NIPAM monomer will be added, and the polymerization of page 12 NIPAM from the reducing end of the oligosaccharides conducted below the LCST of PNIPAM (note that grafting onto cellulose 44 and some non-starch polysaccharides 45 has been achieved successfully using cerium(IV) by other groups, without formation of PNIPAM homopolymer 44 ).

After this grafting step, the reaction mixture will be subsequently heated above the LCST of PNIPAM, to create self-emulsifying nanoparticles. Then a hydrophobic synthetic monomer (MMA) will be added together with a hydrophobic initiator to carry out further polymerization inside the self-emulsifying nanoparticles and grow latex particles. The hydrophobic initiator lauroyl peroxide is chosen in order to minimize polymerization in the aqueous phase. A crosslinker may be introduced during the NIPAM polymerization step, for example to modify the temperature-responsive character of PNIPAM block copolymers. 45 This may help the formation of PNIPAM particles stabilized by grafted oligosaccharides before further polymerization with MMA. Crosslinked PNIPAM-g-oligosaccharides are expected to be loose assemblies of several covalently bound, fully water-soluble, block copolymer species at low temperatures, and to form particles with a condensed PNIPAM core and water-soluble oligosaccharide corona at high temperatures, possibly from more than one of the covalently bound species.

Characterization of the cationic oligosaccharides

In the synthetic pathways used in the present work, the cationized starch Amylofax was cleaved to oligomers by isoamylase or -amylase. The characterization of the original Amylofax and of the oligosaccharides is not trivial, as they are charged, composed of two distinct monomer units, and at least partially branched; indeed, Amylofax is a branched random copolymer. Typical commercial cationized starches, including Amylofax as used in this work, have a high degree of substitution (DS), measured here as 4.8 % (Table 1). Amylofax is expected to have a weightaverage molecular weight, -M w , around 3010 6 , as estimated by aqueous multi-angle laser light scattering size-exclusion chromatography (MALLS SEC, also known as MALLS GPC, gelpermeation chromatography). 20 The -M w of Amylofax is expected to be lower than that of the original potato starch; however, molecular weight values determined by MALLS SEC depend on the specific refractive index increment, dn/dc, 46 and the change of dn/dc before and after page 13 modification of the original potato starch is not known. An increase in dn/dc would lead to increase in the apparent molecular weight determined by SEC. Enzymatic hydrolysis has been studied in the literature by chromatographic techniques, mainly SEC. However, we do not believe that the SEC separation of cationized starch can currently be quantitatively understood using literature data and methods, since the presence of both composition gradients and the branches mean that separation is by size, not by molecular weight, and the presence of charges can lead to adsorption/aggregation effects which currently plague SEC of polyelectrolytes. 47,48 Secondly, no standard is available for cationic starch to calibrate the columns. Thus only potentially misleading apparent molecular weights could be determined by conventional calibration, and multipledetection techniques require an accurate value of dn/dc. Not only is dn/dc unknown for cationized starch but it may also vary with the DS of each polymer chain and may not be constant throughout the molecular weight distribution of the cationized starch. Characterizing Amylofax before and after enzymatic degradation by chromatography is beyond the scope of the present work.

1 H solution-state NMR was used to reliably measure average characteristics of the Amylofax before and after enzymatic degradation. The anomeric 1 H signal comes out at 5.34 ppm for midchain (1→4)- and the non-reducing end, at 5.19 ppm for the  reducing end, at 4.61 ppm for the  reducing end, at 4.94 ppm for the branching point (1→6)-. 49 When the glucose unit is substituted with hydroxypropyl-trimethyl-ammonium, the anomeric 1 H signal comes out at 5.58 ppm and the CH3 signal of the substituent at 3.21 ppm. 50,51 Their respective signal areas are denoted I (1→4)- , I red , I red , I (1→6)- , I sub and I CH 3 . The number-average degree of polymerization of the debranched oligomers, -X n , is then obtained from: 50

-X n = I (1→4)- + I (1→6)- + I sub + I red + I red I red + I red (1)
The degree of branching DB (as % of the glucose units) is then found using the following relation: 50 page 14

DB = 100 I (1→6)- I (1→4)- + I (1→6)- + I sub + I red + I red (2)
The degree of substitution DS (as % of the glucose units substituted by a hydroxypropyltrimethyl-ammonium group) is obtained using the following relation: 52,53 DS = 100

I CH 3 9(I (1→4)- + I (1→6)- + I sub + I red + I red ) (3)
In D2O at 80 C, the signal of the H-2 of the  reducing end is around 3.2 ppm, which overlaps with the CH3 signal. 49,50,54 Thus when the calculation of DS is done on a degraded sample exhibiting a significant number of reducing ends, I CH 3 /9 must be replaced with I CH 3 /9 -Ired in the formula above.

The characterization by 1 H solution-state NMR of Amylofax before and after enzymatic degradation is shown in Figure 1 and Table 1. The original Amylofax shows a DS of 4.8 %, which compares well with the producer's stated value of 4.7 % obtained by the conventional Kjedahl method, 55 and a DB of approximately 3 %. Its -X n was not determined due to insufficient signal from sparse reducing ends. After enzymatic degradation by isoamylase, the strong decrease of the signal at 4.9 ppm indicates extensive debranching, while the appearance of the signals at 5.19 and 4.61 ppm indicates the creation of  and  reducing chain ends. 2 The residual signal at 4.9 ppm indicates incomplete debranching. The isoamylase enzyme was used to debranch the cationized starch using the same protocol as for neutral starch. 8 While this is a well characterized reaction for native starch, it has been observed to be incomplete for cationized starch, as the presence of a cationic substituent close to the branching point affects the activity of the isoamylase enzyme. 50,53 The resulting oligomers have -X n ~ 34 glucose units, 1 to 2 charges per oligomers in average, and 7 % probability of having a branching point. The enzymatic degradation by -amylase does not significantly affect the DS or the DB, as expected for the random cleavage of (1→4)- linkages. The resulting oligomers have -X n ~ 10 glucose units, a 50

% probability of bearing one charge, and a 30 % probability to have a branching point. Both the debranched and randomly cleaved products are good candidates to provide electrosteric page 15 stabilization of latex particles after debranching and grafting, due to their short size and charged character.

Development of a solid-state NMR method for the quantification of grafted starch in a latex

The efficiency of enzymes used for quantifying starch is known to be affected by the presence of synthetic polymer and of cationic substituents on the oligosaccharides. 50,53,56 Furthermore, the centrifugate containing the grafted species was not soluble in any common solvent. 8 Infrared could detect the starch and the synthetic polymer but suffered from too limited a resolution to allow quantification. 44,57 Therefore, it was decided to quantify the grafting efficiency by 13 C solid-state NMR. NMR [START_REF] Claridge | High-resolution NMR techniques in organic chemistry[END_REF][START_REF] Schmidt-Rohr | Multidimensional solid-state NMR and polymers[END_REF] has been used previously to investigate grafted copolymers of starch and synthetic monomers. In solution, 1 H NMR has been used to quantify the starch and synthetic polymer components, [START_REF] Wang | [END_REF] while 13 C NMR has been used to detect them. 60,61 In the solid, 1 H 62 and 13 C 63,64 NMR have both been used to detect the starch and synthetic polymer components.

However, to the best of our knowledge, no quantification of grafting in starch-g-synthetic polymer has been carried out by solid-state NMR before the present work. Tikuisis et al. have used solid-state NMR to quantify starch in starch-polyethylene blends. 65 There are two main methods to record 13 C spectra of solid samples under magic-angle spinning (MAS): single-pulse excitation (SPE-MAS) and cross-polarization (CP-MAS). [START_REF] Schmidt-Rohr | Multidimensional solid-state NMR and polymers[END_REF] SPE-MAS consists in irradiating the 13 C nuclei in the sample with a radio-frequency pulse and recording their resulting signal. As the NMR-active 13 C nuclei represent only 1 % of the C nuclei in natural abundance, SPE-MAS is usually not very sensitive, and requires long acquisition times. CP-MAS comprises irradiating the 1 H nuclei in the sample with a radio-frequency pulse, then transferring the magnetization from 1 H to 13 C nuclei, then recording the 13 C signal. As 99.99 % of the H nuclei are the NMR-active 1 H, CP-MAS is usually more sensitive than SPE-MAS for rigid samples, for which the crosspolarization magnetization transfer from 1 H to 13 C nuclei is efficient.

page 16

Both SPE-MAS and CP-MAS were explored to quantify starch in the grafted species. The final latex obtained by the ab initio synthetic pathway involving PNIPAM (Scheme 2, bottom) was used for method development. 13 C SPE-MAS and CP-MAS spectra of the final dialyzed and ultracentrifuged latex are shown in Figure 2(a): resolved signals are observed at 115 ppm for PNIPAM, at 75, 85, 105 ppm for Amylofax, and at 55 ppm for PMMA (see Figure S3 and S4 for the spectra of pure Amylofax and pure PNIPAM, and the literature [START_REF]NMR spectra database of polymers[END_REF] for the PMMA spectrum).

The CP-MAS spectrum exhibits a higher signal-to-noise ratio than that of the SPE-MAS one. In 2. To estimate the precision of the quantification method, we compare the ratio of the NIPAM-to-MMA quantities in centrifugate and blend independently from the initial weights and from the NMR signal integrals; these values are 0.839 and 0.807 respectively. The quantity of grafted Amylofax oligosaccharides in the final latex, Amylofaxg, is calculated using the NMR signal integrals for PNIPAM and Amylofax page 17

(oligosaccharide), IPNIPMA and IAmylofax, and the initial quantities of NIPAM and Amylofax, NIPAMi and Amylofaxi, according to Equation ( 4):

( ) ( ) ( ) blend PNIPAM Amylofax te centrifuga PNIPAM Amylofax blend i i i g I I I I PNIPAM Amylofax NIPAM Amylofax = (4)
The grafting efficiency GE is then calculated using the following formula:

i g 100 (%) Amylofax Amylofax GE = (5)
The standard deviation SD of a NMR signal integral due to insufficient signal-to-noise ratio SNR is estimated using Equation ( 5): 66

1.28 238 (%) SNR SD = (6)
The error is calculated from the signal-to-noise ratios of the signals involved (PNIPAM and Amylofax, respectively 30 and 40), and Equation ( 6), assuming that the errors are additive. This yields a grafting efficiency of 65 ± 3.5 %. The same calculation carried out using MMA instead of NIPAM yields 52 ± 1.5 %. Although the calculation done using the PMMA signal seems more accurate, care must be taken: this signal must be strongly baseline-corrected to remove the underlying foot of a neighboring intense signal, potentially introducing a large error. Thus we estimate the grafting efficiency to 65 ± 3.5 %. The quantities of PMMA, PNIPAM and grafted oligosaccharide actually present in the final, centrifuged latex are listed in Table 2. These were calculated assuming complete monomer conversion for PNIPAM and PMMA, and Equation ( 5) with 65 % grafting efficiency for Amylofax oligosaccharides.

page 18

Latexes obtained by the different synthetic pathways

Grafting debranched cationized starch onto a seed latex

The synthetic pathway is described on Scheme 2 (top). The debranched Amylofax ( -X n ~ 34 glucose units, 1 to 2 charges per oligomers in average, and a 7 % probability of a branching point) was grafted onto a seed latex of PMMA electrostatically stabilized by DTAB. A stable latex was obtained. A slight increase in latex particle size (from 99 to 106 nm in z-average diameter, measured by HPPS) was observed between the seed and grafted latex. The seed and grafted latex were characterized by TEM (see respectively reference 8 and Figure 3 (a)). Similar particle sizes were observed before and after grafting. Starch was not directly observed in the grafted latex, as the sample was not stained, but it is indirectly detected because the starch-grafted particles deform but do not merge under the electron beam (see arrows). PMMA particles indeed melt under the electron beam, and thus neighboring PMMA particles merge unless another species is present between them and act as physical barrier. 8 Dialysis efficiently removes the electrostatic stabilizers that were used to colloidally stabilize the parent seed latex. 8 To test that colloidal stability was not due to residual added electrostatic stabilizers, the seed latex and the PMMA-g-Amylofax latex were dialyzed. The PMMA seed latex sedimented after 2 days while the PMMA-g-Amylofax, now only colloidally stabilized with grafted oligosaccharides, sedimented after 14 days (Table 3). Thus the oligomers from Amylofax do indeed provide extra stability to the grafted latex. Furthermore, the sedimented grafted latex is redispersable in water simply with moderate hand-shaking, and within experimental uncertainty shows the same particle size before phase separation and after redispersion (Figure 3 glucose sequentially with -amylase and then amyloglucosidase, and quantifying the glucose by spectrometric detection). The efficiency of the kit with pure Amylofax (as opposed to neutral starch) was first measured to be 78 %. This was taken into account when measuring the starch content of the ultracentrifuged latex. Starch amounted to 0.55 wt % of the centrifugate, which corresponds to a grafting efficiency of only 8.5 % of the initial starch. This is an underestimate, as the presence of synthetic polymer and the cationic substituents are known to affect the activity of the -amylase. 50,53,56 A grafting efficiency of 8.5 % is lower that the 33 % previously observed for neutral Amioca starch following the same protocol. 8 This might be due to the ionic character of the oligosaccharides used in the present work, which could lower the entry efficiency in the latex particles. 67 The surface coverage was calculated in terms of latex particle surface area per grafted oligosaccharide chain, following the approach detailed previously. 8 Using the -X n value of 34 determined by NMR, a surface coverage of 24 nm 2 per cationized oligosaccharide chain was determined. This is larger than literature critical values for synthetic PEO macromonomers used as steric stabilizers, 68,69 indicating a high stabilizing potential of the cationized oligosaccharides.

The area per stabilizer is smaller than the value of 103 ± 10 nm 2 obtained for the latex stabilized with debranched neutral starch, 8 and may explain the higher stability (sedimentation after 14 days vs 10 days upon dialysis). The foregoing provides a proof of principle that a stable latex can be obtained by grafting linear charged oligosaccharides onto a synthetic polymer latex, following the same protocol as with oligomers from native starch.

Grafting branched cationic oligosaccharides onto an ab initio latex

In this alternative synthesis pathway (Scheme 2, middle), the degradation of cationic starch to smaller fragments was conducted by random cleavage with -amylase. The oligosaccharides so obtained are branched and can be of various sizes (and thus bear a range of charges). In the present work, the resulting oligomers have -X n greater than 10 glucose units, more than 50 % probability of bearing one charge, and more than 30 % probability of having a branching point (see above). MMA and a CAN solution were added directly to the reaction mixture resulting from the cleavage experiment. A latex was obtained with a solid content of 2 wt %. This exhibits page 20 rather large particle sizes with a mean particle diameter of 260 nm, the same size also being obtained after ultracentrifugation and redispersion. Furthermore, the particle size distribution from TEM is broad (Figure 3 (c-d)). The latex phase separated a few days after dialysis, but could be redispersed by hand-shaking (Table 3): the redispersed latex also exhibited the same particle size as the original dispersion (Figure 3 (c-d)). The amount of charged oligosaccharides actually grafted onto the PMMA particles was determined with the Total Starch Analysis Kit to be at least 2 wt % of the centrifugate, yielding a grafting efficiency of at least 24 % of the initial starch. The particle coverage per oligosaccharide was not calculated in that case, because the mean particle diameter obtained from DLS (see Supporting Information) is a z-average, not the number-average one necessary for the calculation, and the particle size distribution is broad (Figure 3(c-d)), which means that it would be meaningless to make comparative use of such data.

Compared to the seeded experiment carried out previously, this ab initio experiment yielded larger particles (260 vs ca. 100 nm in diameter) and a broader particle size distribution. As expected, the ab initio process is not as controlled as the seeded one, and thus less versatile. More (or less) extensive treatment with -amylase could yield shorter (or longer) oligosaccharides, allowing tailoring of the ab initio latex synthesis. However, as the oligomers are branched and charged, there is no easy way of characterizing them other than by determining average quantities by NMR, and thus this method was not explored further. The observed large particle size and broad particle size distribution are attributed to slow particle nucleation, as detailed below. The observed low entry efficiency of the block-oligomer radical species into latex particles is due to both (a) the low efficiency of the initiation with cerium(IV) and (b) a high critical degree of polymerization of MMA for the amphiphilic block-oligomers to actually form or enter particles. 67 This results in a long particle formation time, and thus in a wide particle size distribution. [START_REF] Gilbert | Emulsion Polymerization: A Mechanistic Approach[END_REF] It also results in a low number of particles, which implies that these particles will be larger (from mass conservation).

The proof of principle has been given that grafting of charged oligosaccharides onto an ab initio synthetic polymer latex is possible following an industrially applicable synthetic pathway.

page 21

An alternative synthetic pathway was investigated to overcome the slow particle nucleation problem.

Grafting branched cationic oligosaccharides onto an ab initio latex avoiding slow nucleation

The synthetic pathway using a comonomer whose corresponding polymer exhibits a LCST is shown in Scheme 2, bottom. The LCST of pure PNIPAM is 32 °C. The LCST of the oligosaccharide-g-oligoNIPAM obtained after the first step of synthesis at 29 °C was investigated by DLS. PNIPAMs grafted to polysaccharides other than starch have been observed to have a LCST only a few °C lower than that of pure PNIPAM. 45 DLS is predominantly sensitive to large species, and thus insensitive to the unimers which are present below the LCST coil-to-globule transition. At low temperatures, the medium is transparent and yellowish, indicating that mainly unimers are present; the intensity detected by DLS was very low. At high temperatures, the medium was turbid, indicating that mostly self-assembling nanoparticles were present, which could consist of a core of precipitated PNIPAM and a shell of covalently bound Amylofax fragments. Their z-average diameter was ~ 130 nm (see Figure S2), the DLS intensity being orders of magnitude higher than that below the LCST. The LCST transition, evidenced by a sharp change in size, occurs between 30 and 40 °C (Figure S2 in the Supporting Information).

Although the cytotoxicity of PNIPAM prevents it use for in vivo medical applications, [START_REF] Twaites | [END_REF]72 these thermoresponsive and double-hydrophilic block copolymers have in themselves a strong potential in other medical applications (as actuator, as artificial muscle or in tissue engineering), to generate thermoresponsive surfaces or in controlled mineralization (as crystal growth modifiers). [73][74][75] Here this material was used as an intermediate.

Heating the oligosaccharide-g-oligoNIPAM to 60 °C and adding MMA and lauroyl peroxide resulted in coagulation of the latex (see Supporting Information). Thus it was decided to add N,N'-methylenebisacrylamide (BIS) to the NIPAM to crosslink the PNIPAM core of the selfemulsifying nanoparticles. BIS is a conventional crosslinker used together with NIPAM, e.g. to page 22

increase the temperature-responsive character of NIPAM-composite materials. 45 The particle size determined by DLS at 60 °C at different steps of the synthesis is shown in Table 4. The oligosaccharide-g-PNIPAM synthesized with BIS exhibited a particle diameter about twice that of the one synthesized in the absence of BIS. This is attributed to crosslinking by BIS, occurring within the particles but also between the particles. There was a further increase in particle size as the crosslinked Amylofax-g-PNIPAM reacts with MMA to form the final product Amylofax-g-(PNIPAM-co-PMMA). There was no significant change in the particle size of the final product before and after dialysis.

The particles obtained before and after dialysis were also examined by TEM (Figure 3(e-f)).

The shape of the particles is not spherical. This is characteristic of PNIPAM particles dried below their LCST, even for crosslinked ones. 76 Dialysis has no significant effect on particle size or morphology.

The grafting efficiency was quantified on the dialyzed and ultracentrifuged latex by solid-state NMR, yielding a grafting efficiency of 65 ± 3.5 % (see above). Note that the concentration in cerium(IV) used here, 35 mmol•L -1 , was higher than the optimal one for grafting PNIPAM onto cellulose, 6 mmol•L -1 . 44 Therefore there may still be room for improving this grafting efficiency.

However, the randomly cleaved Amylofax was extremely efficient as electrosteric stabilizer: the dialyzed latex had not sedimented after 1 year, while all previous latexes had sedimented within a few days or weeks (Table 3).

Comparing the present ab initio pathway with the previous one above, the use of a comonomer whose corresponding polymer has a LCST should theoretically yield smaller particle sizes due to faster nucleation, but unexpectedly it actually yielded larger particle sizes (450 vs 260 nm in diameter). This is ascribed to inter-particle crosslinking by BIS. The grafting efficiency increased from 24 % to 63 %. Even considering the fact that the presence of the synthetic polymer and of cationic substituents may affect the enzymes used to determine the 24 % grafting efficiency previously, this is definitely a significant improvement. Furthermore, the final latex was stable page 23

for over a year. Thus it has been demonstrated that it is possible to graft oligomers obtained by random cleavage of cationic starch onto an ab initio synthetic latex, with a high grafting efficiency, through the use of a comonomer whose corresponding polymer exhibits a LCST.

CONCLUSIONS

Three synthetic pathways were designed to obtain well-characterized synthetic polymer colloids colloidally stabilized by cationized oligosaccharides using cerium(IV), which selectively initiates synthetic monomer polymerization from the reducing end of starch oligomers (grafting from):

seeded, ab initio and ab initio with thermosensitive intermediate. The cationized oligosaccharides were obtained via enzymatic degradation of a cationic starch, either via debranching by isoamylase or via random cleavage by -amylase. A proof that a stable latex can be obtained by grafting linear charged oligosaccharides onto a synthetic polymer latex was demonstrated for each of the three synthetic pathway. Furthermore, a latex that was colloidally stable for more than a year was successfully obtained via the ab initio pathway with a thermosensitive intermediate, using oligosaccharides from random cleavage of a cationized starch by -amylase. The synthetic procedures given here have been devised based on current mechanistic understanding of latex particle formation and growth, and of starch/free-radical chemistry; the fact that these procedures lead to the targeted type of latex supports the underlying mechanistic hypotheses.

A 1 H solution-state NMR method was presented to characterize the original cationized starch and the cationic oligosaccharides in terms of an accurate quantification of their degree of substitution, degree of branching and number-average chain length. We also demonstrated a new method to accurately quantify the starch fragments grafted on the final latex. The 13 C solid-state NMR method developed in the present work is assumption-free and overcomes the limitation of the classical enzymatic methods, which are affected by the presence of synthetic polymers and cationic substituents. 50,53,56 It takes advantage of the fact that the 13 C CP-MAS spectrum of the final latex exhibits resolved signals for the individual components, and can easily be calibrated using a model blend of the individual components.

page 24

There is still room to further optimize those synthetic pathways. One crucial parameter is the size distribution of the oligomers, which could be obtained by size-exclusion chromatography (SEC). The kinetic aspects of the syntheses should be further examined to bring insight into the mechanism of formation of the latexes. [START_REF] Gilbert | Emulsion Polymerization: A Mechanistic Approach[END_REF] These composite materials have potential for large-scale industrial applications such as paper coating and 1-3 soil protection against water erosion 4 (both of which applications would require refining the synthetic procedures to yield higher solids), as well as value-added ones such as drug delivery 5,6 and as models study the biological activity and colloidal stability of living cells. 5,7,8 Their use for in vivo medical applications such as drug delivery 5,6 may be limited by residual cerium, although cerium compounds have been used

successfully in medical applications such as antiemetic and antiseptic in the case of extended burns. 77

Syntheses with PNIPAM and no BIS

The oligosaccharide-g-oligoNIPAM reaction mixture described in experimental part was heated to 60 °C, then 1.92 g of MMA and 0.95 g of lauroyl peroxide (dissolved in the MMA) were added at once. The reaction was left to run overnight; by the next morning, the latex had coagulated. It was thought that feeding the MMA could improve its stability. Consequently, the oligosaccharide-g-oligoNIPAM reaction mixture described in experimental part was heated to 60 °C, then 1.92 g of MMA and 0.95 g of lauroyl peroxide (dissolved in the MMA) were added over 30 min. The reaction was allowed to run for an hour after the end of the feed. Filtering the reaction mixture on large size mesh revealed that there had been no coagulation. 1.2 mL of 1.02 mol•L -1 trisodium citrate was S-4 added to the reaction mixture, which was then neutralized with NaOH. On neutralization, a significant amount of a yellowish precipitate was observed. It was surmised that since the starch is cationic, neutralizing it leads to its loss of efficiency as a colloidal stabilizer. However, samples taken from the reaction mixture (and not neutralized) also coagulated. When those samples were filtered, it was noticed that the remaining liquid phase underwent a transition from slightly cloudy to an intense cloudiness when heated to 60 ºC. This suggests that some of the oligoNIPAM chains were not tightly bound to the particles.

In order to trap the oligoNIPAM chains within the particles, the same protocol was followed, except that N,N-methylenebisacrylamide (BIS) was added (at 10 mol % of NIPAM), and the reaction was done at 60 ºC. After 1.5 h the system coagulated. On cooling down it then formed a gel. The gel formation can be explained as follows. At 60 °C, the radical flux is very high and radical formation is not only at the reducing end of the oligosaccharide but also along its chain. In the presence of BIS, it then becomes easy to form a cross-linked network. Ideally the cross-linking should occur inside the particles. It was thought that reacting the NIPAM and BIS at 29 ºC and then raising the temperature to 60 ºC would lower the radical flux and allow the BIS to form crosslinks within the particles before adding MMA. The BIS concentration was also lowered to 5 mol% NIPAM. This last synthesis is developed in the main text.

S-5 13 C solid-state NMR spectra of individual components and model blend

  particular, SPE-MAS does not detect the Amylofax and PNIPAM signals between70 and 120 ppm in 15 h, while CP-MAS detects them with a high signal-to-noise ratio in 9 h. Therefore CP-MAS was chosen to quantify the amount of Amylofax actually present in the centrifugate. CP-MAS is not a quantitative technique, as different sample parts may exhibit different crosspolarization transfer efficiencies. However, it is possible to calibrate the CP-MAS measurement of the centrifugate by measuring a blend of the same components in similar ratios under the same conditions. This allows the determination of correction factors that can then be applied to the signal integrals of the different components measured on the CP-MAS spectrum of the actual centrifugate. Note that all components will have the same NMR response in both cases (in particular, they will have the same relaxation behavior), as the NMR experimental parameters are exactly the same, and only a negligible proportion of each component is close from a covalent link to another component in the grafted sample. Thus our CP-MAS experiment calibrated by a CP-MAS measurement of a model blend constitutes an assumption-free method to directly quantify oligosaccharides in oligosaccharide-g-copolymer samples.A zoom of the region of interest of the baseline-corrected CP-MAS spectrum of the centrifugate is shown in Figure2(b). Accurate values of the component weights and of the integrals of the signals of interest are detailed in Table

  (b)). After one year storage the latex was still redispersable. The sedimentation is surmised to originate in insufficient coverage of the latex particles by the oligomers (see below). The quantity of Amylofax anchored on the resulting latex was evaluated by subjecting the ultracentrifuged latex to the Total Starch Analysis kit (Megazyme) following the Total Starch Assay Procedure AOAC method 996.11 (which comprises enzymatically digesting the starch to page 19

FIGURES Scheme 1 . 31 Scheme 2 . 32 Figure 1 .

 1312321 FIGURES

Figure 2 : 34 Figure 3 .

 2343 Figure 2: 13 C solid-state NMR spectra of the final dialyzed oligosaccharide-g-(PNIPMA-co-PMMA) (a) SPE-MAS (14 h 45 min) vs CP-MAS (9 h), (b) baseline-corrected spectrum used for quantification with signal assignment.

Figure S1 .

 S1 Figure S1. The intensity distributions for dynamic light scattering measurements of Amylofax in

Figure S3: 13 C

 13 Figure S3: 13 C CP-MAS spectrum of pure Amylofax.

Figure S4: 13 C 6 Figure

 136 Figure S4: 13 C CP-MAS spectrum of pure PNIPAM.

Table 1 .

 1 Number-average chain length -X

	TABLES
	Sample	-X n	DB (%) DS (%)
	original	not determined	2.8	4.8
	debranched	34	0.4	4.6
	randomly cleaved	10	3.2	5.0
	Figure 1.

n , degree of branching DB and degree of substitution DS of original Amylofax, Amylofax after debranching by isoamylase and Amylofax after random cleavage by -amylase. Values were calculated using Equations (

1

) to (3) on spectra shown on

Table 2 .

 2 Component quantities and signal integrals on the CP-MAS spectra of the final dialyzed

	respectively. The NMR signals are integrated over 49.5-62 ppm for PMMA, 64-88 ppm for
	Amylofax, 110-122 ppm for PNIPAM.

oligosaccharide-g-(PNIPAM-co-PMMA) and the model blend. mblend, minitial and mcentrifugate are the initial quantities used in the model blend, in the grafting experiment, and the final quantities present in the final, centrifuged latex respectively; Iblend and Icentrifugate are the integrals of the NMR signals in the CP-MAS spectrum of the model blend and of the final, centrifuged latex,

Table 3 .

 3 Characteristics of the oligosaccharide-g-polymer latexes obtained by the different synthetic pathways described on Scheme 2. a underestimate by enzymatic method; b by solid-state NMR; c not determined because the high polydispersity of the latex means that it would be meaningless to make comparative use of such data.

	Latex	seeded	ab initio	ab initio
		PMMA-g-Amylofax	PMMA-g-Amylofax	(PMMA-co-PNIPAM)-g-Amylofax
	Oligosaccharides	debranched	randomly cleaved	randomly cleaved
	Grafting	8.5 % a	24 % a	65  3.5 % b
	efficiency
	Mean particle	106 nm	260 nm	450 nm
	diameter
	Surface coverage	24 nm 2	nd c	nd c
	/ oligosaccharide
	Stability	14 days, redispersable	few days,	> 1 year
		for > 1 year	redispersable

Table 4 .

 4 Particle sizes obtained as z-average diameter by DLS at different steps of the synthesis with BIS following Scheme 2 (bottom). 9000AT EN correlator. The sample temperature was controlled by heating decalin in the void volume. The intensity measured in DLS varies as the 6 th power of the radius; it is thus extremely sensitive to large particles. Only experimental runs with less than 500 kilocounts per second on average were used. Any features in a size distribution produced by dynamic light scattering (also known as PCS, photon correlation spectroscopy) software must be tested for artifacts by varying the basic assumptions as to the form of the distribution (e.g. CONTIN vs NLLS2,3 ) and numerical parameters in the inversion such as integration range. Only results for which both CONTIN and NLLS deconvolution routines gave qualitatively similar results in terms of number and maxima of size populations in the intensity distribution were considered. The measurements were done with 0.1 wt% Amylofax in deionized water, at 80 °C and 90° angle. The following values were used: refractive index 1.32287, viscosity 0.3547 mPas. No species of diameter above 220 nm was detected, even after just a few minutes in solution (FigureS1); this seems to indicate a fast

	S-1

ACKNOWLEDGMENTS

The support of Discovery, LIEF, and Linkage grants of the Australian Research Council is gratefully acknowledged. Chu-Wai Wayne Yuen is thanked for the dynamic light scattering measurement, Jonathan Peate and Ashton Reeve for a debranching experiment, Dirk-Jan Voorn for helpful comments on the manuscript. The collaboration of the University of Sydney Electron Microscope Unit with transmission electron microscopy is acknowledged. M.G. thanks the University of Queensland Centre for Magnetic Resonance for the use of the solution-state and solid-state NMR equipment.

SUPPORTING INFORMATION AVAILABLE

Additional methods as well as results and discussion.


Hank De Bruyn

Ewan Sprong

Marianne Gaborieau

John A Roper

Robert G Gilbert
email: b.gilbert@uq.edu.au

Starch-graft-(synthetic copolymer) latexes initiated with Ce 4+ and stabilized by amylopectin

Keywords: colloids, dispersions, emulsion polymerization, graft copolymers, polysaccharides

A method is presented for synthesizing surfactant-free latexes comprising starch-graft-(vinyl polymer) starting with a suspension of amylopectin, either native or modified, then using cerium(IV) with either potassium persulfate or glucose to create grafting sites on the starch. Latex particles comprising polystyrene, poly(styrene-co-n-butyl acrylate) and poly(vinyl acetate) grafted onto high molecular weight amylopectin were developed, with up to 80 % of the starch effectively grafted to the particles. These latexes were colloidally stable against electrolyte (several months in 4 M NaCl). Reaction rates of Ce 4+ with simple sugars and polysaccharides

INTRODUCTION

There are significant potential uses of starch-graft-poly(vinyl) copolymer latexes, with applications such as improvements over the current technology of blending starch suspensions with synthetic polymer latexes, commonly used in papermaking. [START_REF] Maurer | Starch and Starch Products in Surface Sizing and Paper Coating[END_REF][START_REF] Maurer | [END_REF] Two limitations of current technologies are as follows. The first is that starch is usually only incorporated in relatively small quantities, primarily as a stabilizer. The latexes so obtained do not benefit as much as one would like from the economies and environmental benefits of greater starch incorporation. The second is that the starch is usually thinned to the point that it loses much of the mechanical strength desirable in many coating applications. This thinning appears to be necessary to avoid gelation of the latex under experimental conditions used in the past. The present paper extends previous work, based on ozone grafting/initiation, 3 to a Ce 4+ -based method for producing stable synthetic polymer latexes with a high proportion of high molecular weight starch grafted to the latex particles.

In our recent paper 3 a method was presented that overcame the gelation problem (the "stabilized precursor" mechanism). Briefly, gelation was ascribed to coalescence of synthetic polymer particles grafted to adjacent amylopectin molecules. It was shown that gelation was prevented if the precursor particles were sufficiently stabilized by the charged end groups of entering oligomers that had been initiated with potassium persulfate. The result was a "scaffold" structure, as sketched in Figure 1, with regions of synthetic polymer "decorating" a starch scaffold, the starch and synthetic polymer being attached by grafting. In this work it will be seen that the same result is achieved when the precursor particles are sterically stabilized by glucose end groups, where glucose was used as an intermediary in the initiation process. Modifications of this synthetic method are explored in this work, as is the mechanism of gelation and the dependence on starch concentration. Ce 4+ has replaced the ozone used previously to create free-radical grafting sites on the starch backbone in combination with potassium persulfate or glucose to enhance the performance of the Ce 4+ and simultaneously stabilize the precursor particles. Ce 4+ oxidation is a commonly utilized industrial technique for grafting synthetic polymer onto polysaccharides such as starch and cellulose. 4,[START_REF] Hebeish | The chemistry and technology of cellulosic copolymers[END_REF] Ce 4+ is known to oxidize many organic substrates such as alcohols and amides to produce the corresponding oxygen-or nitrogen-centered radicals. [START_REF] Moad | The Chemistry of Free Radical Polymerization 2nd Edn[END_REF] It is found to complex and react rapidly with 1,2-diols causing cleavage and, it is thought, production of a radical at the cleavage site. [START_REF] Moad | The Chemistry of Free Radical Polymerization 2nd Edn[END_REF][START_REF] Mcdowell | [END_REF] The 1,2-diols in starch are trans to one another and somewhat sterically hindered. As such, they may not be as easily complexed to Ce 4+ as straightchain 1,2-diols and therefore perhaps not as readily cleaved. However, it is well known that Ce 4+ creates free radical graft sites on starch and cellulose, primarily at C2 and C3 carbons. [START_REF] Moad | The Chemistry of Free Radical Polymerization 2nd Edn[END_REF]8,9 Molecular modeling studies suggest that, following β-scission, alkyl radicals may be formed on C1, C2, C3 and C4 carbons on the main chain. 10 Another interesting reaction of Ce 4+ with starch takes place at the hemiacetal "reducing end" of the starch molecule, where the reaction with Ce 4+ is much faster than at the 1,2-diols in the body of the starch chains. 8,10 Contrary to our previous work using oligosaccharides, 11 the starches used in this paper are amylopectins with molecular weights of the order of 10 7 to 10 8 . As there is only one "reducing end" per starch molecule and 10 5 to 10 6 anhydroglucose units in amylopectin, it follows that most radical sites formed by direct Ce 4+ oxidation of amylopectin will be in the body of the starch chains. Reaction rates of Ce 4+ with simple sugars and polysaccharides are investigated here, as described below.

Creation of free-radical grafting sites on the starch backbone is facilitated by an interesting reaction pathway whereby glucose (which reacts extremely rapidly with Ce 4+ ) can be used as an intermediary to transfer radical activity to the starch. 12 Glucose radicals so formed can also initiate homopolymerization, thereby generating aqueous-phase glucose-terminated styrene oligomers. These oligomers, which would be expected to follow classical Maxwell-Morrison entry kinetics (describing the rate and mechanism of entry of radicals into latex particles in emulsion polymerizations), 13 might enter precursor particles in sufficient numbers to stabilize the precursor particles against coalescence. This point is discussed further below.

Finally, Ce 3+ is oxidized to Ce 4+ by potassium persulfate-indeed this is the method commonly used to produce cerium(IV) salts. The cerium(IV)/persulfate binary system has been used to graft acrylamide onto starch. 14 The oxidation of Ce 3+ by persulfate is enhanced by catalytic amounts of silver(I). 15 Skarzewski 16 used the cerium/persulfate/silver ternary system to oxidize various substrates, and although they found that it worked very well for oxidizing naphthalenes to quinines, oxygen-containing substrates produced low yields. They attributed this to complexing of Ce 4+ to alcohols and their consequent removal from the oxidation system. However, in the case of starch, the 1,2-diols reduce the Ce 4+ and return Ce 3+ to the catalytic cycle. This may be a useful pathway to explore in future as a means of reducing the amount of the relatively expensive cerium salts required for industrial scale grafting.

EXPERIMENTAL

Materials

The monomers used (styrene, Huntsman; n-butyl acrylate (BA) and vinyl acetate (VAc), Aldrich) had their inhibitors removed on alumina columns and were stored at 4 °C for not more than a few days before use. Monomers were purged with nitrogen for 15 min immediately prior to use. Iron(II) sulfate (Univar, 99.0 %), cerium(IV) ammonium nitrate (CAN, Univar, 99.99 %) and potassium persulfate (KPS, Merck), were all used as supplied.

Waxy corn starches (Mazaca, a native amylopectin kindly provided by Penfords) or StacKOTE™ 7, a modified amylopectin (kindly provided by National Starch) were gelatinized in a Siltex 175D autoclave (30 min at 120 °C) in sodium acetate buffer (pH 3.5, 0.1 M) for all experiments. The molarity of starch suspensions is given as concentration of anhydroglucose (AHG) units throughout. D-(+)-Maltose monohydrate (> 99 %) was purchased from Sigma. Sodium acetate buffer (pH 3.5, 0.1 to 0.2 M) was prepared from sodium acetate (Merck, pro analysi) and glacial acetic acid (Ajax Finechem, analytic reagent grade) used as supplied. Milli-Q water (18.2 MΩ⋅cm -1 ) was used throughout. All latex syntheses were conducted under a nitrogen atmosphere.

Syntheses

Reactions were undertaken in a 1 L Pyrex water-jacketed reactor equipped with an overhead stirrer, and ports (with septa) for the addition of reagents.

Reactions of Ce 4+ /Ce 3+ with sugars and starch 0.5 mL of maltose or Mazaca solution or suspension was added to 3.0 mL of 0.01 M CAN in 0.1 M H 2 SO 4 at 60°C in a UV/Vis cuvette to give a slight excess of saccharide. The absorbance at 420 nm was monitored and reaction half-lives determined.

Effect of (poly)saccharides and Ce 3+ on persulfate decomposition

Glucose or starch (0.1 M) was added to a solution of KPS (0.01 M) with and without Ce 3+ and maintained at 50 °C or 70 °C. Excess iron(II) was added to oxidize any unreacted KPS and the resulting iron(III) back-titrated with Ce 4+ according to the procedure of Vogel. 17

Enzymatic digestion

Approximately 5 mL of latex was diluted to 20 mL, adjusted to pH 6.5 with sodium hydroxide, 2 drops of α-amylase were added and kept at 75 °C overnight.

Gelation studies

Latexes 1 to 6: A series of starch-g-poly(BA-co-styrene) latexes were prepared on a 500 mL scale using Ce 4+ as initiator with 1, 2, 3, 4, 5 and 6 wt% Mazaca, a constant starch-to-synthetic monomer ratio of 1:1.8, and a monomer mixture with 50/50 w/w of styrene/BA. Samples were taken during the reaction for each synthesis. Typically, a Mazaca suspension of the required concentration was prepared in acetate buffer (0.01 M, pH 3.5) and autoclaved at 121 °C for 30 min. The Mazaca suspension so obtained was heated to 80 ˚C, after which was added 1 mL of 2 mM CAN solution and 0.2 mL of 235 mM KPS solution for each gram of Mazaca. This was followed immediately with addition of monomer (1.8 g for each gram of Mazaca) and KPS (1 mL of 235 mM KPS solution for each gram of Mazaca) fed over 50 min. The reaction was maintained at 80 ˚C for a further 4 h. Final pH values ranged from 1.4 to 2.1.

Stable latexes

Latex 7: cerium(IV)/persulfate/modified amylopectin/poly(vinyl acetate). Gelatinized StacKOTE™ 7 (600 g, 1 M) was heated to 80 °C and purged with nitrogen for 15 min. KPS (5 mL, 0.123 M) and CAN (1.0 mL, 0.018 M) solutions (made fresh immediately before use) were added as single shots. 10 mL of the same KPS solution was co-added (at 20 mL•h -1 , 30 min) with VAc (20 mL at 20 mL•h -1 , 60 min). The KPS/VAc addition was repeated. The reaction was cooled to 60 °C, VAc was added (100 mL, 25mL•h -1 , 4 h) and kept at 60 °C overnight.

Latex 8: cerium(IV)/glucose/native amylopectin/styrene. A 10 wt% Mazaca suspension was prepared in the usual manner (autoclave, 121 °C, 30 min). To 165 g of the 10 % Mazaca suspension (0.09 mol AHG units) was added 1.6 mmol CAN solution (10.36 mL, 0.155 M at 0.065 mL⋅min -1 for 159 min), 1.6 mmol glucose (6.67 mL, 0.242 M, at 0.042 mL⋅min -1 for 159 min) and 95 mmol styrene (8.83 mL at 0.055 mL⋅min -1 for 161 min). The mixture was stirred under nitrogen in a round-bottom flask at 60 °C overnight. Residual monomer was postpolymerized with 0.09 g KPS in 10 mL of water at 60 °C.

Latex 9: cerium(IV)/glucose/modified amylopectin/styrene/BA. This was similar to Latex 8 but using StacKOTE™ 7 instead of Mazaca and adding some 50/50 w/w styrene/BA respectively in a second stage monomer feed to give a final wt% composition: starch 14.8 %; styrene 10 % and BA 7 %.

Step 1: A 17 wt% StacKOTE™ 7 suspension was prepared in the usual manner (autoclave, 121°C, 30 min). To 750 g of the 17 wt% StacKOTE™ 7 solution at 60 °C was added CAN solution (0.29 M at 0.173 mL⋅min -1 for 160 min), glucose solution (0.74 M at 0.0857 mL•min -1 for 160 min) and styrene (30 mL at 0.273 mL•min -1 for 110 min). The mixture was stirred under nitrogen in a round-bottom flask at 50 °C overnight (sample 9A).

Step 2: The temperature was raised to 60 °C and CAN solution (0.29 M at 0.133 mL•min -1 for 45 min), glucose solution (0.74 M at 0.067 mL•min -1 for 45 min) and styrene/BA 50/50 (w/w) (1.0 mL⋅min -1 for 45 min) sample 9B. Step 2 was repeated twice to give samples 9C and 9D.

Finally, potassium persulfate (0.27 g) was added and the temperature held at 70°C for two hours to give samples 9E.

CHARACTERIZATION

Solid content

The determination of the solid content of the latexes 1 to 5 was carried out by gravimetry after 1 night at 60 ºC.

Viscosity measurements

Viscosity measurements were conducted on latexes 1 to 5 using a Brookfield DVII viscometer and an appropriate spindle, at 100 rpm.

Transmission electron microscopy

Samples were dried a carbon-coated copper grid and analyzed using a Biofilter Philips CM120 Transmission Electron Microscope with a CCD camera and an accelerating voltage of 120 kV.

Grafting efficiency

For the present purposes, starch grafting efficiency is defined as the percentage of initial starch grafted to final latex particles. This was measured by ultracentrifugation of latex (90,000 rpm for 30 min), with determination of the starch content of the supernatant by gravimetry. Blanks were performed on starch suspensions; it was found that a very small amount of a brownish impurity precipitated but this was insignificant in terms of mass balances.

Size Exclusion Chromatography (SEC)

Samples taken at different times during the synthesis of latex 9 were ultracentrifuged (1 h at 90,000 rpm) and the supernatant was analyzed by SEC. The conditions for the SEC analysis were the following: Shimadzu LC-10ATVP pump, Shimadzu DGU-14AVP degasser, Shimadzu SIL-10ADVP autoinjector, Shimadzu CT10ACVP column oven, Wyatt Optilab DSP interferometric refractometer; Waters columns: Ultrahydrogel guard column + Ultrahydrogel 500; eluent: 0.05 M ammonium acetate set at pH 5.2 with glacial acetic acid + 0.05 % sodium azide; flow 0.5 mL⋅min -1 .

RESULTS AND DISCUSSION

Reactions of Ce 4+ /Ce 3+ with saccharides Ce 4+ was reduced by Mazaca (equimolar in AHG units), the reaction having a half-life of 250 s at 60 °C and 125 s at 80 °C. As expected, this was much slower than the reaction of Ce 4+ with maltose (t ½ ~ 10 s at 60 °C). However it is fast enough that is clearly necessary to feed the Ce 4+ continuously (latex 7) or regenerate it with an oxidizing agent such as KPS (latexes 8 and 9) when reactions are carried out over several hours.

Effect of saccharides and Ce 3+ on persulfate decomposition

Decomposition of KPS at 50 °C induced by saccharides was found to be extremely rapid in the presence of glucose and only slightly less so in the presence of StacKOTE TM 7 (Table 1).

Decomposition of persulfate at 70 °C in the presence of Mazaca was also much faster than normal thermal decomposition in absence of Mazaca (Table 1). Surprisingly, the presence of Ce 3+ reduced the apparent decomposition rate of persulfate in both glucose and Mazaca solutions/suspensions but this may be due to some unanticipated side reaction in the titration used to determine the unreacted KPS. Blank experiments conducted without KPS indicated that (1) the presence of starch has no effect on the titration and (2) Ce 3+ is oxidized by KPS under these conditions. Most likely the Ce 3+ is oxidized to cerium(IV) by sulfate radicals and persists in that form long enough to affect the titration. The Ce 3+ itself does not appear to reduce persulfate, at least not at a significant rate. Rather, persulfate decomposes thermally or via a one-electron oxidation of another species (e.g. silver(I) 15 or in this case starch) to produce a sulfate ion and a sulfate radical. It is this sulfate radical that oxidizes Ce 3+ to Ce 4+ .

Gelation studies

Latexes 1 to 6: Latexes 1 to 5, based on 1 to 5 wt% Mazaca, all reached a high conversion soon after the end of the monomer feed stage (50 min) with solids content increasing slightly thereafter (Figure 2). Similarly, viscosities approached their maxima shortly after the monomer feed ended (Figure 2). Final viscosities (10 to 200 cP for 1 to 5 wt% starch suspensions respectively) were much higher than that of starch suspensions of the same concentrations, indicating a trend toward gelation. "Latex" 6, with 6 wt% starch, resulted in a gel and was not further characterized.

Latexes 1 to 5 were ultrasonicated (Sonics Vibracell, 30 %, 5 min), after which their viscosities reduced to approximately that of the equivalent raw starch suspensions (5 to 30 cP for 1 to 5 wt% starch suspensions respectively). This is strong evidence of the "scaffold" structure of the latex and the associated gelation mechanism described briefly above and in detail in our previous paper. This series of latexes showed that, if no precaution is taken to avoid coalescence of the grafted precursor particles, gelation of amylopectin-based latexes occurs when the concentration of amylopectin exceeds 5 wt %, which is approximately the overlap concentration c* for amylopectin. [START_REF] Perry | [END_REF] It should be noted in this regard that the entry efficiency (entry of oligomeric radicals into particles 13 ) in this system is probably low, due mainly to the very low water solubility of monomers, and hence stabilization of precursors by sulfate end groups is minimal. The intended role of KPS in this system is to regenerate Ce 4+ from Ce 3+ continuously during the reaction, not to provide electrostatic stabilization to the precursor particles. The limitation of low entry efficiency is overcome, for example in latex 7, by using VAc/KPS, which is expected to exhibit almost 100% entry efficiency 13 and thereby confers electrostatic stabilization to the precursor particles.

Stable latexes

Latexes 7 to 9 are stable latexes with a high proportion of starch (10 to 15 % by total weight depending on the solubility of the starch) and a high proportion of that starch grafted to latex particles; these have been produced using the following procedures. The three presented below are typical. The procedure is versatile and robust and is by no means limited to the examples below.

Latex 7 was synthesized from starch and vinyl acetate using CAN and KPS for initiation. It exhibited a final solids content was 21.7 %, and comprised 9.7 % polyVAc and 12.0 % starch (of which 48.7 % was grafted). The final latex was stable against dialysis and sedimented after treatment with α-amylase (starch digesting enzyme). This behavior demonstrated that the final latex was stabilized primarily by grafted starch. It formed a translucent film which did not redisperse in water.

Latex 8 was synthesized from starch and styrene, using CAN and glucose for initiation. Although the latex appeared quite thick during the first three hours, it did not gel. After postpolymerization of the residual monomer with KPS, it looked like an ordinary latex. It is proposed that gelation was avoided by the "stabilized precursor" mechanism described above, with glucose as the stabilizing moiety. A grafting efficiency of 60 % was achieved. The particle sizes in the resulting latex were small and polydisperse, as exemplified by the TEM micrograph in Figure 3. It is interesting to note that KPS added in the final step grafted more starch onto the latex particles (78.4 % compared to 68.3 % before addition of KPS). This could be due to regeneration of Ce 4+ or direct grafting by KPS.

CONCLUSIONS

A method has been developed whereby highly branched starch molecules in solution (amylopectin or modified amylopectin) can be induced to act as a scaffold to form latexes with a synthetic polymer using free-radical polymerization. The procedure is versatile and robust and has been demonstrated to work with diverse monomer systems (vinyl acetate, styrene/butyl acrylate) and initiation systems (cerium/glucose, cerium/persulfate). The Ce 4+ /Ce 3+ couple creates radical sites on the amylopectin chains which then act as macro-initiators for free-radical polymerization in emulsion. Surfactant-free, starch-graft-copolymer latexes can be made based on amylopectin from native waxy starches, with minimal degradation if the precursor particles are stabilized against coalescence at early times in the reaction.

A potential benefit of this approach is that most of the starch is grafted to synthetic latex particles and as such is unable to migrate away from the hydrophobic film formed on the substrate surface in wet applications such as paper coatings.

Figure 1 .

 1 Figure 1. Sketch of the scaffold structure wherein regions of synthetic polymer (round shapes) are grafted to a starch molecule.

3

 3

Figure 2 . 5 (

 25 Figure 2. Evolution of (a) solid content and (b) viscosity with time for latexes 1 to 5 (squares: latex 1, circles: latex 2, triangles: latex 3, diamonds: latex 4, stars: latex 5).

Figure 3 :

 3 Figure 3: TEM micrograph of final Latex 8.

Figure 4 :

 4 Figure 4: Evolution of the chromatogram of the non-grafted starch with reaction time for latex 9. Traces A to D correspond to different reaction times (see experimental section), trace E to after the post-polymerization with KPS, and unreacted StacKOTE™ 7 is shown for comparison.

Table 1 : Induced decomposition of persulfate by saccharides (KPS 0.01 M;

 1

	saccharide 0.1 M)
	saccharide		decomposition
	nil	3 h at 50 °C	6 %
	Glucose	3 h at 50°C	36 %
	StacKOTE™ 7	3 h at 50 °C	30 %
	nil	1 h at 70 °C	11 %
	Mazaca	1 h at 70 °C	65 %

ACKNOWLEDGEMENTS

The support of a Linkage Grant from the Australian Research Council and The Dow Chemical Company, Midland Michigan 49674 USA, are gratefully acknowledged. The Key Centre for Polymer Colloids was established and supported under the Australian Research Council's Research Centres program. The University of Sydney Electron Microscope Units is gratefully acknowledged for its support. We greatly appreciate interactions with Dr Brian Hawkett and Dr Paul Perry.

GRAPHICAL ABSTRACT

A method is presented for synthesizing surfactant-free latexes comprising starch-graft-(vinyl polymer) using Ce 4+ to create grafting sites on the starch. Colloidally stable polystyrene, poly(styrene-co-n-butyl acrylate) and poly(vinyl acetate) latexes grafted onto high molecular weight amylopectin were developed. Furthermore, reaction rates of Ce 4+ with simple and polysaccharides were investigated, as well as gelation mechanism of the latex.

starch monomer

Ce 4+