Synthesis of functionalized nanoparticles via copolymerization in microemulsions and surface reactions

Reactive & Functional Polymers 33 (1997) 49-59

REACTIVE 84 FUNCTIONAL POLYMERS

Synthesis of functionalized nanoparticles via copolymerization in microemulsions and surface reactions Chantal Larpent a,*, Elisabeth Bernard a, Joel Richard b*l, Sophie Vaslin b aS.I.R.C.O.B.,

EP CNRS 102, Universiie de Versailles-Saint Quentin en Yvelines, 45 Avenue des Etats-Unis, 78035 Versailles, Cedex, France b Rh&e-Poulenc Recherches, 52 Rue de la Haie Coq, 93308 Aubervilliers, Cedex, France

Received 10 December 1996; revised version received 22 January 1997; accepted 22 January 1997

Abstract Oil-in-water microemulsions of mixtures of styrene and comonomer are easily prepared using titration methods in the presence of nonionic (NPn) or anionic (SDS) surfactants. Functionalized nanoparticles in the 20-30-nm diameter range bearing chloromethyl, active-ester, acid or pyridyl surface end-groups are prepared by polymerization of microemulsions containing mixture of styrene (St) and, respectively, vinylbenzylchloride (VBC), N-acryloyloxysuccinimide (NHA), methacrylic acid (MA) or vinylpyridine (VP). Reactions of nucleopbiles on particles bearing either chloromethyl or active-ester surface end-groups, performed directly in the aqueous suspensions, give rise to a wide range of nanoparticles with various functionalities. The main role of the sut-factant on such surface reactions is demonstrated and used to improve the reaction yields. Keywords: Polymerization in microemulsion; Functionalized nanoparticles; Surface reactions; Chloromethyl; Active-ester surface end-group

1. Introduction Aqueous suspensions of nanoparticles may find very specific applications in drug delivery, microencapsulation, biomedical diagnosis [l-3] provided that suitable ligands or binding groups are linked to the surface to ensure recognition. Moreover, the very large specific area of particles in the 20-30-nm diameter range offer new opportunities in other domains like catalysis and chro-

*Corresponding

author.

’Permanent address: Centre de microencapsulation, 8 rue A. Bloquel, 49100 Angers, France.

matography. Therefore, there is a great demand for highly functionalized nanoparticles bearing ‘at will’ various kinds of chemical functions. Aqueous suspensions of nanoparticles can be obtained by polymerization of hydrophobic monomers in oil-in-water microemulsions and numerous studies have been devoted to polymerization in microemulsion during the last decade [4,5]. Nevertheless, the preparation of highly functionalized nanoparticles via copolymerization in microemulsion has been scarcely studied [l&10] and no general procedure for their synthesis has been proposed. In this paper, we describe a versatile method leading, in one or two steps, to highly function-

1381-5148/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PIIS1381-5148(97)00015-l

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C. Larpent et al. /Reactive & Functional Polymers 33 (1997) 49-59

alized nanoparticles with various functionalities. Actually, one of the most important limitations for performing copolymerization in microemulsion is the preparation of the starting microemulsion: the addition of a new component most often modifies the phase diagram and therefore the composition of the microemulsion domain. We show that microemulsions of mixture of monomers are easily prepared using titration processes. The polymerization of these microemulsions leads to nanoparticles bearing various kinds of chemical functions. The synthesis of well-defined highly functionalized nanoparticles can also be achieved by performing surface modifications of previously prepared nanoparticles: this route is more versatile than copolymerization, provided that suitable reactive groups are linked to the surface. To the best of our knowledge, such chemical modifications have never been performed on nanosized particles prepared by polymerization in microemulsion although one can obviously take advantage of the high specific area resulting from the reduced size. Nevertheless, nucleophilic substitutions on polychloromethylstyrene microspheres or crosslinked resins have been successfully used to introduce various chemical functions via reaction of nucleophilic anions or amines [l l-131. Reactions of primary amines on polymer-bearing N-hydroxysuccinimide ester groups have been used to perform chemical modifications [14,15]. We show that such chemical reactions can be performed on nanoparticles bearing chloromethyl or active-ester surface end-groups leading to a wide range of functionalized nanoparticles. Moreover, we demonstrate that one can take advantage of the ‘microemulsion process’ to control the level of functionality. 2. Results and discussion 2.1. Copolymerization in microemulsions Two simple and versatile methods, based on titration, have been used to prepare oil-in-water microemulsions of mixture of two monomers.

The first method, developed by Tadros et al., [16], uses a mixture of two nonionic surfactants: this consists in forming a water-in-oil emulsion using a low-HLB surfactant (NP5) and then titrating with an aqueous solution of a high-HLB surfactant (NP15). The second one, widely described, uses a mixture of an ionic surfactant and a cosurfactant [17-201 and consists in titrating an o/w emulsion, stabilized using an anionic surfactant (sodium dodecylsulfate: SDS), with an alcohol (pentanol) until a clear microemulsion is obtained. These titration processes readily permit the adjustment of the microemulsion composition. In the present work, both of these methods have been found successful for preparing o/w microemulsions of various mixtures of styrene (St) and functionalized comonomers like vinylbenzylchloride (VBC), N-acryloyloxysuccinimide (NHA), vinylpyridine (VP) and methacrylic acid (MA) with molar ratio styrene/comonomer ranging from 90/10 to 70/30 (Table 1 and Scheme 1). Polymerizations of these microemulsions have been performed under mild conditions below 35°C in order to ensure the microemulsions remain stable during the reaction and to avoid side reactions on functional groups. Water-soluble redox systems like hydrogen peroxide/ascorbic acid at 30°C [ 161 and ammonium persulfate/tetramethyldiaminomethane at room temperature [21] have been mainly used to initiate the polymerizations. An oil-soluble radical initiator, DMPA (2,2-dimethoxy-2_phenylacetophenone), decomposed under UV irradiation has also been successfully used [22]. Whatever the experimental conditions, polymerization of both monomers, monitored by gas chromatography, readily takes place, reaching 100% conversion within 2 h (Table 1). It can be seen that stable translucent bluish suspensions of nanoparticles with diameter in the region of 20-30 nm are obtained with both types of microemulsion provided that the radical initiator is properly chosen. These suspensions are remarkably stable since neither flocculation nor aggregation of the particles have been observed after more than one year.

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51

& Functional Polymers 33 (1997) 49-59

Table 1 Copolymerization in microemulsions No.

Monomers a (molar ratio)

wt% b

Surfactant a

Init. c,d

D (nm)’

Polymer comuosition f

Surface functions s

PI P2 P3 P4 P5 P6 P7 P8 PQ

StfVBC (85/15) St+VBC (85/15) St+NHA (80/20) St+NHA (85/15) StfNHA (85/15) StfVP (QO/lO) StfVP (80/20) St+MA (75125) StSMA (85/15)

5.6 7.5 6.1 6.4 6.4 8 7.9 4.7 5.6

NPn SDS SDS SDS SDS NPn SDS NPn SDS

I1 11 12 I3 I2 Ilh Ilh 12’ 12’

27 rt 2 25rt2 20&2 21f2 23 f 1 20 f 5 30f4 17f3 20 f 3

87113 86114 80120 85/15 85/15 QO/lO 84/16 85/15 84/16

10.80 meq/gj (~70%) >0.85 meq/gJ (?70%) 1.020 meq/g (42%) 1.200 meq/g (70%) 0.785 meq/g (46%) 0.480 meq/g (50%) 0.750 meq/g (40%) 0.235 meq/g (20%)

a See Scheme 1 and text for abbreviations. b Monomers wt% in the starting microemulsion. c Radical initiator; 11: (NH4)zSzOs/TMDAM, 20°C; 12: HzOzlascorbic acid, 30-35°C; 13: DMPAAJV, 20°C. d Reaction time: 2 h. e Particle diameter determined by QELS and TEM. f Wcomonomer molar ratio in the polymer determined from elemental analysis. s Eq. surface functions/g polymer, in brackets: eq. surface functions/eq. functional unit in the polymer (in %), h Unstable suspensions of larger particles are obtained when initiating system I2 is used. i Larger particles with a broad distribution are obtained when initiating system 11 is used. j Determined from the maximum substitution yields (Table 2).

2.2. Characterization of the polymers

ence of methacrylic acid units is demonstrated by the carbonyl stretching band at 1725 cm-’ in the IR spectra. In the case of nanoparticles containing MA or VP, the amount of acid and pyridyl surface endgroups, determined by conductimetry and acidbase reaction, range from 15 to 50% of the total amount of functional units in the polymer. For styrene-NHA particles, the amount of surface active-ester groups has been determined using the chemical method described for hydrophilic gels [14] by allowing the particles to react with etbylamine and measuring the UV absorbance due to N-hydroxysuccinimide (NHS) (Eq. 1). The amount of surface active-ester groups is much higher on nanoparticles resulting from DMPA-initiated polymerization (P4, 70%) than

Elemental analysis and spectroscopic studies of the resulting polymers, isolated by flocculation, demonstrate that the comonomer has been incorporated in the polymer with a molar ratio styrenekomonomer close to that expected on the basis of the composition of the monomer mixture used in its preparation. In the case of styrene-NHA copolymers, the IR spectra show the characteristic imide vibration bands at 1782 cm-’ and 1811 cm-’ (Fig. la) and the ‘H- and 13C-NMR spectra exhibit the metbylene and carbony1 signals expected for the succinimide unit. The ‘H- and 13C-NMR spectra of styrene-VP copolymers show the characteristic pyridyl resonances. For styrene-MA copolymers, the presWI)

9

9 C-O-N

6

3

0 St-NHA

+

RNH2

-

C-NHR k

+

HO-N 3 0 NHS

C. Larpent et al. /Reactive & Functional Polymers 33 (1997) 49-59

52

Surfactants

W-h

%H25SQNa

O(CH2CH20)nH

NPn

[NP5: n=5 ; NPls : n=15]

SDS

Comonomers

0

/ co)-/

CH3 +

N

C02H

MA

VP

VBC

Reactants WJU-b)20H Ethanolamine : EA

WWWdW Hexanediamine: HDA

Ph-CH-CH-NH2 bH

WdzN-b

I=Hs

Norephedrine : Neph

H2N(CH&S03Na Taurine : Tau

R-CH,

,W C02H

R=CH,:

Aminoethylpyridine : AEP

Ala

R=CH,Ph:

Phe

HN’NH HO&OH

H3C

H

H3C

H2N

Glucosamine : GA

4-aminotempo : AT

Biotine Hydrazide : BH

Scheme 1.

on nanoparticles resulting from hydrogen peroxide/ascorbic acid-initiated polymerization (P3, 42%). These results demonstrate that the level of surface functions is directly related to the location of the radical initiator and can be easily modified by the proper choice of the initiating system. Initiator-dependent mechanism of polymerization must obviously account for such an effect. Although further kinetic studies should be performed for this peculiar copolymerization, it

is noteworthy that such mechanistic discriminations have already been observed for copolymerization of styrene with polymerizable cosurfactants [lo]. 2.3. Su$ace reactions: nucleophilic substitution of chloromethyl sur$ace end-group Chloromethylated nanoparticles prepared by copolymerization of styrene and VBC in mi-

C. Larpenc et al. /Reactive & Functional Polymers 33 (1997) 49-59

53

a

Fig. 1. IR spectra of St-NHA copolymer before and after reaction with norephedrine. (a) Poly St-NHA. (b) Polymer resulting from reaction of norephedrine on poly St-NHA.

C. Larpent et al. /Reactive

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& Functional Polymers 33 (1997) 49-59

Table 2 Surface reactions on particles bearing chloromethyl groups a No.

Suspension b (surfactant)

Reactant

No. eq. e

Yield * (%)

Bound reactant (mmol/g) s

Db (nm)

Pl-1 P2-1 Pl-ld P2-ld Pl-2 P2-2 Pl-3 P2-3 Pl-4 P2-5a P2-5b P2-5d P2-6a P2-6b P2-7 P2-8 P2-9 Pl-10 P2-10

Pl (NPn) P2 (SDS) Pl (NPn) dialyzed’ P2 (SDS) Pl (NPn) P2 (SDS) Pl (NPn) P2 (SDS) Pl (NPn) P2 (SDS) P2 (SDS) P2 (SDS) dialyzed d I’2 (SDS) F’2 (SDS) P2 (SDS) P2 (SDS) P2 (SDS) Pl (NPn) P2 (SDS)

KSCN i KSCN i KSCN ’ KSCN i NazSOs i NazSOs i MeNH2 j MeNH2 j

12 12 12 12 12 12 9 9 9 12 2 2 12 2 2 2 2

71 40 57 5 35 13 36 68 30 64 51 25

0.83 0.48 0.66 0.06 0.40 0.16 0.41 0.83 0.35 0.78 0.69 0.30 0.86 0.49 0.76 0.47 0.37

28f4

EA j AEP j AEP j

AEPj Ala Alaj Nephj Tauj HDA j

NaOH k NaOH k

10

40 62 38 70 51 40

18&3

31 ??4 27 Zt 3

18f4 28f3 19f5 19f4 17f3

a See Scheme 1 for abbreviations; all reactions were performed at 30°C for 48 h. b Starting suspensions: Pl (persulfate/diamine-initiated polymerization in nonionic microemulsion, initial particle diameter 27 nm), P2 (persulate/diamine-initiated polymerization in ionic microemulsion, initial particle diameter 25 nm). c Reaction performed after partial removal (about 90%) of the NPn surfactanta by dialysis. d Reaction performed after removal (>98%) of the surfactant (SDS) by dialysis. e No. eq.: mole nucleophile/eq. chlorine in the polymer. *Substitution yield calculated from the total amount of chlorine in the polymer. s Amount of reactant in the final polymer (from elemental analysis). h Diameter of the resulting particles determined by QELS. i pH-7. j pH-10-12. k pH-12.

croemulsions have been reacted at 30°C with various nucleophiles (Table 2, Scheme 1). The reactions were performed directly on the aqueous suspensions obtained by polymerization of microemulsions formulated either with nonionic (NPn) or anionic (SDS) surfactants. The results given in Table 2 demonstrate that the substitution of the chloromethyl surface groups occurs with various nucleophiles like anions (sulfite or thiocyanate), primary amines and polyfunctional amino-ligands like aminoalcohols or aminoacids. Various chemical groups can thus be linked to the particles surface. Moreover, the particles size and distribution remain almost unchanged after surface modifications (Table 2). The substitution yields, calculated from the

total amount of chlorine in the starting polymer, range from 10% to about 70% depending both on the nucleophilic reactant and on the surfactant. Thus, with anionic nucleophiles, the substitution yields are higher in suspensions containing nonionic surfactants than in suspensions containing anionic surfactants: respectively 70% and 40% for potassium thiocyanate (Pl-1, P2-1) and 35% and 13% for sodium sulfite (PI-2, P2-2). Electrostatic repulsions between the anionic surfactant adsorbed on the particles and the anionic reactant may account for the lower yields in suspensions containing SDS. On the other hand, reaction with amines occurs with higher yields in suspensions containing the anionic surfactant (P2-3, Pl-3). As shown by blank experiments (Pl-10, P2-IO),

C. Larpent et al. /Reactive & Functional Polymers 33 (1997) 49-59

hydrolysis of the chloromethyl group by hydroxy anions becomes a competitive side reaction in basic medium; owing to the above-mentioned electrostatic repulsions, the presence of anionic surfactant molecules adsorbed on the particles hinders the hydrolysis side reaction resulting in higher substitution yields with neutral amino nucleophiles. It is notable that a decrease of the pH value reduces the hydrolysis reaction [ 131: thus, in the presence of nonionic surfactants, it would be preferable to lower the pH value to about 8 to preserve the highest number of reactive chloromethyl surface groups and to ensure highest amino-ligand linkage. However, some reactions have been performed in suspensions after removal of the surfactant by dialysis (partial removal for NPn and almost complete removal for SDS): whatever the nucleophile, the substitution yields are always much higher in the presence of surfactant than after removal of the surfactant (Pl-l/PI-ld, P2-l/P2-ld, P2-5ab/P2-5d). The wetting of the particle surface and the decrease of the interfacial solidliquid tension may account for this tremendous effect of the surfactant concentration on our reactions involving a water-soluble reactant and a fairly hydrophobic particle surface. The influence of the hydrophilicity of the reactive groups microenvironment has already been observed for reactions of water-soluble reactants on chloromethylated crosslinked polymers [ 111. Nevertheless, it is noteworthy that the amount of surfactant in the starting suspensions (-8% wt SDS; -12% wt NPn) is far above the cmc and that micelles are formed outside the polymer particles. From a practical point of view, one can thus take advantage of the above-mentioned surfactant-effect to get the highest substitution yields by the proper choice of the starting microemulsion. However, if surfactant-free functionalized nanoparticles are required it is highly preferable to remove the surfactant after the chemical modification.

55

2.4. Su$ace reactions: reaction of amino-ligands on active-ester surface end-group Styrene-NHA nanoparticles bearing active esters, prepared by DMPA or (hydrogen peroxide/ascorbic acid)-initiated copolymerizations of styrene and NHA in anionic microemulsions, have been reacted at pH=7.5 with various amines (Table 3, Scheme 1). Suspensions resulting from SDS-based microemulsions have been used in order to limit the expected hydrolysis of the active esters [14,15]: elemental analysis and blank experiments show that hydrolysis does not compete significantly at pH=7.5 (only 5% of surface groups). Surface reactions, monitored by UV absorption of N-hydroxysuccinimide liberated in situ (Eq. 1) [14], occur with fairly good yields (60 to 100%) whatever the level of surface functionalization (suspensions P3 and P4, Table 3). The chemical modifications of the polymer are confirmed by elemental analysis and by the disappearance of the succinimide bands at 1782 and 1811 cm-’ in IR spectra (Fig. lb). The surface reactions can be achieved with low molar excess or sub-stoichiometric amount of nucleophile and take place with simple amines as well as with polyfunctional amino-ligands. The poor nucleophilicity of the hydrazine function may account for the slightly lower yield. Moreover, the surface reactions do not modify either the particle size or the colloidal stabili145.33ty (Table 3). Various amino-ligands including amino-alcohol, amino-acid, amino-sugar, amino-TEMPO and biotine hydrazide can thus be linked to the nanoparticles with a surface excess allowing further biomedical or chemical applications. 3. Conclusion Polymerization in microemulsions followed by surface reactions thus allows the synthesis of a wide range of functionalized nanoparticles with diameter in the region of 20-30 nm. Further investigations are currently being developed into the utilization of the very large specific area of such particles for chemical or biomedical applications.

C. Larpent et al. /Reactive & Functional Polymers 33 (1997) 49-59

56

Table 3 Surface reactions on particles bearing N-hydroxysuccinimid ester groups a No.

Starting suspension b

Reactant

No. eq. c

Yield % d

Bound amino-ligand (mmol/g)

P3-la P3-lb P4- 1 P3-2 P4-2 P3-3 P3-4 P3-5 P4-6

P3 P3 P4 P3 P4 P3 P3 P3 P4

EtNHz EtNH2 EtNH2 Neph Neph Phe GA AT BH

1000 1.5 1.5 1.5 1.5 1.5 1.5 0.6 0.11

100 (42) 100 (42) 100 (70) 100 (42) 100 (70) 70 (30) 60 (25) 60 (40) f 5 (3.5) s

1.00 1.00 1.20 0.95 1.10 0.71 0.61 0.72 0.06

DC (nm)

. 23 f 3 20*2 25 !c 3

28f4 23 & 3

a See Scheme 1 for abbreviations; reactions performed in HEPES buffer (pH=7.5) for l-4 days. b Starting suspension: P3 (H202/ascorbic-acid-initiated polymerization, initial particle diameter 20 nm), P4 (DMPA-initiated polymerization, initial particle diameter 21 nm). c No. eq.: mole nucleophile/eq. NHA equivalents in the polymer. d Substitution yield vs. surface ester groups; in brackets: substitution yield vs. total amount of NHA in the polymer. e Diameter of ihe resulting particles determined by QELS. f 100% yield/amine. s 76% yieldlhydrazine.

4. Experimental section 4. I. Materials and methods Synperonic NP15 and NP5 (nonylphenolethoxylates with respectively about 15 and 5 ethoxy units), sodium dodecylsulfate (SDS, 99%) were supplied by ICI and Janssen respectively. Commercial monomers, radical initiators and reactants were used without further purification. Styrene was distilled before use. N-Acryloyloxysuccinimide (NHA) has been prepared according to the previously described procedure [ 141. The infra-red spectra were recorded using a FTIR apparatus (Nicolet 250) on polymer film obtained by slow evaporation of dilute chloroformic solutions. The NMR spectra were recorded on a Brucker AM300 spectrometer, ‘H (300 MHz) and 13C (75.5 MHz); the 13C nuclei were attributed from 13C{ 1H} and DEPT spectra. Elemental analysis have been obtained from the Service Central d’Analyse (CNRS, Vernaison, France). Quasi-elastic light scattering (QELS) analysis was obtained with a 3W Spectra Physics laser (Brookhaven BI 2030 correlator), Centre de Recherches d' Aubervilliers, Rhone-Poulenc, France. Some samples were analysed by transmis-

sion electron microscopy (TEM, obtained from the Centre de Microscopic, Universite de Rennes I, France), the photographs are in good agreement with the QELS results. Gas chromatography analysis were performed on a Carlo Erba FTV4000 equipped with a RSL 150 (25 m) capillary column. The samples were prepared according to the previously described procedure [lo]. 4.2. Microemulsionspreparation Microemulsions with nonionic surfactants: a 2-wt% aqueous solution of sodium dodecylbenzenesulfonate (2 g) is added to a mixture of monomers (styrene and comonomer, 7 g) and NP5 (1 g) giving rise to a milky emulsion upon magnetic stirring. A 15-wt% aqueous solution of NP15 is then gradually added until a transparent microemulsion of low viscosity is formed. Microemulsions with SDS/pentanol: the desired amount of monomers (styrene/comonomer, typically 3 g) is added to a IO-wt% aqueous solution of SDS (typically 45 g); magnetic stirring of the resulting mixture leads to a milky emulsion which is gradually titrated with pentan-l-01 until a transparent microemulsion of low viscosity is obtained.

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4.3. Polymerization

& Functional Polymers 33 (1997) 49-59

reactions

4.3.1. Water-soluble radical initiators The previously prepared microemulsion is transferred into a three necked round bottom flask and degassed with nitrogen for 20 min. The two components of the desired redox couple are then successively introduced with a syringe. When the H202/ascorbic acid system [10,16] is used, hydrogen peroxide (30 wt% in water, 0.15 mol/mol monomer) and then ascorbic acid (aqueous solution 12.5 g l-l, 0.03 mol/mol monomer) are added at room temperature and the reaction mixture is then heated under nitrogen at 3035°C with an oil bath for 2 h (100% conversion, GC analysis). When the ammonium persulfate/tetramethyldiaminomethane system [ 10,2 I] is used, ammonium persulfate (0.02 mol/mol monomer) in the minimum amount of water and then pure tetramethyldiaminomethane (0.05 mol/mol monomer) are successively added to the microemulsion. The reaction is then carried out at room temperature (20-25°C) under nitrogen for 2 h (100% conversion, GC analysis). 4.3.2. Oil-soluble radical initiator [10,22] 2,2-Dimethoxy-2-phenylacetophenone DMPA (0.007 mol/mol monomer) is solubilized in the monomer blend before the preparation of the microemulsion. The freshly prepared microemulsion is transferred into a reactor equipped with a UV lamp and degassed with nitrogen for 20 min before the lamp is switched on. The reaction is carried out at room temperature (20-25”(Z) under nitrogen for 3 h (100% conversion, GC analysis). 4.4. Separation and characterization resulting polymers

of the

The polymers were separated after flocculation according to the previously described procedure [lo]. For spectroscopic and elemental analysis, the polymers were further purified as follows: 500 mg of the previously isolated polymer is dispersed in 100 ml acetone and the suspension stirred for at least 2 h. The polymer, separated

57

by filtration, is solubilized in chloroform and the resulting solution dried over magnesium sulfate. The pure polymer, isolated after removal of the solvent under reduced pressure, is then characterized by elemental analysis and IR and NMR spectroscopy when soluble. 4.4. I. Spectroscopic data of the copolymers Poly St-VP: ‘H NMR (CDC13, 6 ppm): 1.44 (broad, CH2); 1.78 (broad, CH); 6.51 (d broad, ArH); 6.72 (broad, Aul/2 = 55 Hz, ArH); 8.31 (broad, PyH). 13C NMR (CDC13, 6 ppm): 40.63 (d broad, ’&-_H = 132 Hz, skeleton CH St); 42.60 (d broad, ’&-_H = 130 Hz, skeleton CH VP); 45.85 to 5 1.15 (numerous CH2 skeleton); 120.67 (d broad, ’&-_H = 169 Hz, Cg Py); 123.34 (d broad, ‘JC-_H = 165 Hz, Cg Py); 125.63 (d broad, ‘JC-_H = 165 Hz, Co St); 127.96 (d broad, ’&-_H = 160 Hz, Cp and Cm St); 135.35 (d broad, ‘JC-_H = 165 Hz, Cy Py), 145.33 (broad, Cipso St.); 149.18 (d broad; JC_H’l70 Hz, Ca! Py); 164.35 (broad, Cipso Py). Poly St-NHA: ’H NMR (CDC13,6 ppm): 1.44 (broad, skeleton CH2); 1.83 (broad, skeleton CH and CH2); 2.66 (broad, CH$O, NHA); 6.57 (broad, ArH); 7.05 (broad, ArH). 13C NMR (CDC13, 6 ppm): 25.60 (sharp, CH2, NHA); 40.41 (broad, skeleton CH); 41.88 to 46.43 (numerous CH2 skeleton); 125.67, 127.69 and 127.98 (broad, Co, Cp and Cm St); 168.81, 168.99 and 169.24 (CO imide, NHA); 171.12, 171.21 and 171.28 (CO ester, NHA). IR (film, u cm-‘): 3050-3000 (strong, CH St), 2900-2850 (strong, CH skeleton), 18 10 and 1780 (CO imide, NHA), 1740 (CO ester, NHA). Poly St-MA: IR (film, u cm-‘): 3050-3000 (strong, CH St), 2900-2850 (strong, CH skeleton), 1725 (CO acid, MA). Poly St-VBC: IR (nujol, LJcm-‘): 3050-3000 (strong, CH St), 2900-2850 (strong, CH skeleton), 1270 (m, CH2Cl). 4.4.2. Determination of sugace groups The determination of the amount of acid surface groups (St/MA particles) was performed by conductimetric titrations. The amount of pyridyl

58

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& Functional Polymers 33 (1997) 49-59

surface groups was determined from the wt% Cl incorporated in the polymer after treatment of the particles suspension with hydrochloric acid, flocculation and purification of the resulting polymer [lo]. The assays for the active esters were performed both on the flocculated polymer and directly on the suspension. In the former case, the flocculated St/NHA copolymer, extensively washed with water, is dried under vacuum and dispersed (40 mg) in water (5 ml). A 35~1 aliquot of the resulting suspension is added to 1200 ~1 of HEPES buffer (0.1 M, pH=7.5), 35 ~1 of 1 M ethylamine solution and 7 ~1 of 1 M mercaptoethanol solution. The mixture is allowed to react under magnetic stirring for 2 days (no further improvements for longer reaction times) and then centrifuged. The amount of N-hydroxysuccinimide (NHS) is determined spectrophotometrically by absorbance at 260 nm [14]. Similar results (f50 peqlg) are obtained when assays are performed directly on the particles suspension according to the following procedure: a mixture containing 1 g of suspension and 600 ~1 ethylamine in 50 ml HEPES buffer is allowed to react for 4 days. A 10-g aliquot is then diluted in 100 ml methanol, the polymer which flocculates upon heating is discarded and the amount of NHS in the filtrate is determined by spectrophotometry (see above). 4.5. S&ace

reactions on particles bearing chloromethyl groups

The reactions have been performed directly on the suspensions prepared by persulfate/diamine-initiated polymerization of StVBC (85/15) in microemulsions formulated with nonionic surfactants (suspension Pl) or with SDWpentanol (suspension P2). The starting suspensions have the following characteristics: Pl: particle diameter 27 & 2 nm; polymer composition, %C: 87.06, %H: 7.46, %Cl: 4.11. P2: particle diameter 25 f 3 nm; polymer composition, %C: 85.24, %H: 7.5 1, %Cl: 4.34. The desired amount of nucleophile (number of equivalent calculated from the total amount

of chloride in the starting polymer) is added to 20 ml of suspension and the reaction mixture is magnetically stirred at 30°C for 48 h. The polymer is then flocculated according to the general procedure described above and the reaction yield calculated from the polymer composition (elemental analysis). 4.6. Sur$ace reactions on particles bearing N-hydroxysuccinimide

ester groups

The reactions have been performed on suspensions prepared respectively by H2Oa/absorbic acid (P3) and DMPA-initiated (P4) polymerization of St-NHA (85/15) in microemulsions formulated with SDS/pentanol. The starting suspensions have the following characteristics: P3: particle diameter 20 f 2 nm; polymer composition, %C: 80.56, %H: 7.21, %N: 1.35, %0: 8.17; 42% (1.02 meq/g) of ester groups at the surface. P4: particle diameter 21 f 2 nm; polymer composition, %C: 78.44, %H: 7.25, %N: 1.69, %0: 10.13; 70% (1.2 meq/g) of ester groups at the surface. 1 g of suspension is diluted in 30 ml HEPES buffer (0.1 M, pH 7.5) and the desired amount of amine (number of equivalents calculated from the total content of ester groups in the polymer) is added. The reaction is stirred for 1 to 4 days at room temperature. 100 ml of methanol are then added and the flocculated polymer is separated by filtration and washed with methanol. The amount of N-hydroxysuccinimide released during the reaction is determined by UV absorption of the combined filtrates according to the procedure describe above. The reaction yields have also been calculated from elemental analysis of the flocculated polymer. When norephedrine and phenylalanine are used, the amount of unreacted amine is determined by HPLC analysis of the filtrates.

Acknowledgements Prolabo S.A. is gratefully financial support.

acknowledged

for

C. Larpeni et al. /Reactive

& Functional Polymers 33 (1997) 49-59

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