Emulsifying Properties Of Biodegradable Polylactide-Grafted Dextran

1014

Biomacromolecules 2008, 9, 1014–1021

Emulsifying Properties Of Biodegradable Polylactide-Grafted Dextran Copolymers J. Raynaud, B. Choquenet, E. Marie, E. Dellacherie, C. Nouvel, J.-L. Six, and A. Durand* Laboratoire de Chimie Physique Macromoléculaire, UMR 7568, CNRS-Nancy-University, ENSIC, BP 20451, 54001 Nancy Cedex, France Received October 4, 2007; Revised Manuscript Received December 4, 2007

Amphiphilic glycopolymers, polylactide-grafted dextran copolymers (Dex-g-PLA), were synthesized with a wellcontrolled architecture obtained through a three-step procedure: partial silylation of the dextran hydroxyl groups, ring-opening polymerization of D,L-lactide initiated from remaining hydroxyl groups, silylether deprotection under very mild conditions. Depending on their proportion in polylactide (PLA), these copolymers exhibited solubility either in water or in organic solvents. The emulsifying properties of these glycopolymers were studied: depending on their PLA-to-dextran ratio, they were able to stabilize either direct or inverse emulsions. Droplet size was related to the amount of amphiphilic copolymer in the continuous phase. The aging mechanism of both direct and inverse emulsions was shown to be Ostwald ripening in the first weeks following preparation. Finally inverse miniemulsion copolymerization of acrylamide and N,N′-methylenebisacrylamide was performed in the presence of an amphiphilic Dex-g-PLA stabilizer. Polyacrylamide hydrogel nanoparticles were prepared in that way.

1. Introduction Recently, with increasing environmental concerns, biodegradable and/or biocompatible materials have become of interest from standpoints of surfactant or biomedical applications. For ecological purposes, polysaccharide-based materials (glycopolymers) are interesting mainly because polysaccharides are obtained from renewable resources and are easily degraded or even biodegradable. Since the pioneering work of Landoll,1 glycopolymer surfactants have been widely studied as emulsifying agents. These hydrophobic polysaccharide derivatives have been mainly obtained by modification of the polysaccharides. The first ones were cellulose derivatives like methylethylcellulose ether or sodium carboxymethylcellulose,1,2 then appeared other derivatives from pullulan,3 alginate,4,5 chitosan,6 inulin,7 or dextran,8–10 for example. Some of these modified polysaccharides present good performance in lowering surface tension and thickening aqueous phase. For several years, our laboratory has been studying these properties in the case of polysaccharidebased surfactants.8–13 Some of us intensively studied the emulsifying properties of dextrans substituted by aliphatic or aromatic groups and recently reported on the links between their emulsifying ability and their structural parameters.14–16 In addition, these stabilizers have been used as polymeric surfactants in miniemulsion polymerization and their potential to prepare stable suspensions of nanoparticles for drug delivery has been reported.17 Contrary to the hydrophobically modified polysaccharides, completely biodegradable glycopolymers have been less studied in terms of surfactant activity or thickening properties although they were recently employed for the potential formulation of hydrogels18,19 or of particles20,21 used as drug delivery vehicles. To the best of our knowledge, the surfactant properties of totally biodegradable polysaccharide-based copolymers such as polylactide-grafted dextrans (Dex-g-PLA) have not been published except from our recent works.22 In addition, few of the reported * Corresponding author. E-mail: [email protected]. Telephone: 33(0)3-83-17-52-92. Fax: 33(0)3-83-37-99-77.

amphiphilic grafted polysaccharide exhibited a well-defined structure. Recently, the controlled synthesis of amphiphilic Dexg-PLA by using a three-step strategy has been reported (Scheme 1).23 On the basis of the ”grafting from” concept, the proposed synthetic pathway involves a partial reversible protection of the polysaccharide hydroxyl functions, the ring-opening polymerization (ROP) of D,L-lactide promoted by all the remaining OH groups of the partially protected dextran, and finally a deprotection step.23–25 The kinetics of hydrolysis of these copolymers were also studied, and prevailing mechanisms were suggested.26 The general aim of this work was to evaluate the emulsifying ability of these totally degradable Dex-g-PLA. We wanted to investigate how the emulsifying properties can be controlled by changing the structural characteristics of the polymeric surfactant and more particularly the length of the PLA grafts. We tried to compare the droplet size and stability of the obtained emulsions to those of emulsions prepared in the presence of other hydrophobically modified dextrans carrying simple hexyl chains. Starting from these results, droplets stabilized by these graft copolymers were used as nanoreactors in an inverse miniemulsion polymerization to produce hydrogels nanoparticles. Inverse miniemulsion polymerization is indeed a convenient method to prepare hydrophilic nanoparticles.27 Although miniemulsion polymerization is now a rather well-documented process, only a few studies involve polymeric stabilizers.28–33 To the best of our knowledge, the use of a biocompatible and degradable copolymer based on polysaccharide as a stabilizer of inverse systems was never reported. Furthermore; hydrogel micro- and nanoparticles are interesting candidates for drug delivery applications.34 They are mechanically more stable than liposomes and exhibit a greater loading capacity. Most of them are sensitive to external stimuli such as pH, temperature, or ionic strength.35 In this work, we focused on polyacrylamide (PAAm) nanoparticles. Such nanoparticles were studied because of the potential pH sensitivity of PAAm-based gels.36–38 Applications of such sensitive polymers as drug delivery systems39,40 or oxygen carriers have already been mentioned.41,42

10.1021/bm701101n CCC: $40.75  2008 American Chemical Society Published on Web 02/14/2008

Biodegradable Polylactide-Grafted Dextran Copolymers

Biomacromolecules, Vol. 9, No. 3, 2008 1015

Scheme 1. Synthetic Pathway to Dex-g-PLA

First, the synthesis and the macromolecular parameter control of these Dex-g-PLA glycopolymers will be resumed, then their solubility properties will be presented. Investigations on the emulsifying properties of these Dex-g-PLA for direct or inverse emulsions will be reported as well as their use as stabilizers of inverse miniemulsions to prepare nanoparticles of polyacrylamide networks.

2. Experimental Section 2.1. Materials. Dextran T40 (Mn ) 33000 g mol-1 and PDI ) 1.26 as characterized by size exclusion chromatography) was purchased from Pharmacia Biotech and dried under reduced pressure at 100 °C for one night. 1,1,1,3,3,3-hexamethyldisilazane (HMDS) (99.9%), chlorotrimethylsilane (TMSCl) (99%), Saccharin, and stannous octoate (SnOct2) were purchased from Aldrich and used without any further purification. After dilution in dry toluene, SnOct2 solution was stored in glass ampules under nitrogen. D,L-lactide from Lancaster was recrystallized twice with dry toluene and dried under vacuum before use. Before use, all solvents were dried and distilled with appropriate methods. 2.2. Dexn-g-yPLAm Synthesis. The whole controlled synthesis of Dex-g-PLA and the characterization of their macromolecular parameters were performed as previously described.23–25 In the following text, the symbol Dexn-g-yPLAm is used to design PLA-grafted dextrans. n and m (in g/mol) are the Mn of the dextran backbone (determined after

deprotection of silylated dextran under mild conditions24) and of PLA grafts (Mngraft). y is the average number of PLA grafts per 100 glucose units. 2.3. Surface Tension Measurements. Surface (water-air) tension measurements were carried out at 25 °C using a K8 surface tensiometer (Krüss, Germany) and applying the Wilhemy technique. All samples were equilibrated for a sufficient time (15 min to 1 h) to reach constant readings. 2.4. Emulsification and Emulsion Composition. Direct and inverse emulsions were prepared by sonication (pulsed mode, 10 W, 2 min) using a Vibracell model 600W (Sonics & Materials Inc., Danbury, CT). Before sonication, the two phases were mixed by application of a vortex stirrer during 1 min. Direct emulsions were dispersions of various oils (decane, dodecane) in aqueous solutions of amphiphilic copolymers. Inverse emulsions were composed of aqueous solutions of salts (NaCl, Na2CO3, Na2SO4) or glucose dispersed in a glycerol triacetate solution of Dex-g-PLA. The volume of the continuous phase was kept equal to 10 mL for all the prepared emulsions. The amphiphilic glycopolymers were previously dissolved in the continuous phase during 20 h. Because of experimental requirements (dilution, duration of size measurements), the so-called “initial average droplet size” of emulsions is in fact the average droplet size determined about 15 min after the beginning of the emulsification step. This fact is not a serious limitation for the oils used in this work.

1016 Biomacromolecules, Vol. 9, No. 3, 2008

Raynaud et al.

Table 1. Macromolecular Parameters of Dexn-g-yPLAma entry

copolymers

Mn (g/mol)b

yc

Mngraft (g/mol)d

FPLAe

1 2 3 4f

Dex33K-g-36PLA2.3K Dex33K-g-24PLA0.9K Dex33K-g-39PLA0.1K Dex29K-g-14PLA0.2K

33000 33000 33000 29000

36 (73) 24 (49) 39 (79) 14 (25)

2300 900 144 200

0.83 0.56 0.25 0.15

a n and m (in g/mol) are the Mn of the dextran backbone and of PLA grafts (also named Mngraft), respectively, and y the average number of PLA grafts per 100 glucose units. b Mn of dextran backbone determined after deprotection of partially silylated dextran24 under mild conditions. c Average number of PLA grafts per 100 glucose units according to ref 23. The mean number of PLA grafts per macromolecules is given in brackets. d Calculated from Mngraft ) 144 × DPngraft. DPngraft was evaluated from method described in ref 23. e PLA weight fraction.23 f The synthesis of this copolymer was reported in ref 23.

2.5. Nanoparticle Preparation by Inverse Miniemulsion. Dexg-PLA was dissolved in glycerol triacetate at various concentrations. The aqueous dispersed phase was composed of a 0.5 M sodium sulfate solution of acrylamide and bisacrylamide (3 vol % related to acrylamide). After stirring for 1 h, emulsification was achieved via sonication. To avoid polymerization due to heating, the mixture was ice-cooled during sonication. Azobisisobutyronitrile (AIBN, 3 wt % of acrylamide) was added in the medium, and polymerization was performed at 65 °C for 2 h. 2.6. Nanoparticle Characterization. Droplets and particle sizes were measured by dynamic light scattering at low concentration using a HPPS-ET from Malvern. The reported diameters were the so-called z-average diameters from cumulant analysis. Three consecutive series of measurements were accumulated, leading to total measurement times of about 5 min. Although this apparatus is able to measure relatively concentrated samples, the emulsions and latexes were diluted. Direct emulsions were diluted with MilliQ water, whereas filtrated glycerol triacetate (previously saturated with water) was used in the case of inverse emulsions. For sizes below 500 nm, the values should be considered to be given with an uncertainty of (5 nm. For sizes between 500 and 900 nm, the experimental uncertainty is (10 nm. Finally, the values above 900 nm should be considered as good orders of magnitude.

3. Results and Discussion 3.1. Synthesis and Surface Active Properties of Amphiphilic Dextran Derivatives. 3.1.1. Synthesis of Polylactide-Grafted Dextran Copolymers (Dexn-g-yPLAm). According to previous detailed studies,23–25,43 new Dex-g-PLA copolymers were designed to study their emulsifying capacities. The macromolecular parameters of these selected copolymers are shown in Table 1: PLA weight fraction (FPLA), average number of PLA grafts per 100 glucose units (y), and lengths of both backbone and grafts. To ensure a controlled architecture to these glycopolymers, they were synthesized through a threestep strategy described in Scheme 1. Hydrophobization of dextran was achieved by a controlled silylation reaction, i.e., leading to a partially silylated polysaccharide with an average number of residual OH functions along the dextran chain. Depending on experimental conditions, high silylation yields could be achieved without any degradation of the polysaccharide backbone.24 Thus, in this work, we used nondegrading conditions for the silylation step (use of saccharin or TMSCl as catalyst, addition of cosolvent like of toluene if necessary) as proved in our previous paper.24 As each of these residual OH is potentially available for the next ROP of D,Llactide and will initiate a graft, the first step allows indeed the control of the average number (y) of PLA grafts per 100 glucose units (Table 1). The objective was to obtain copolymers with moderate number of grafts per chain but with various grafts lengths.

Figure 1. Chemical structure of hydrophobically modified dextrans with 1,2-epoxyoctane. In this figure, the hydrophobic substituent is located on one position in the sugar ring but the detailed study of the position modified by the aliphatic epoxide (among the three possible ones) has not been performed yet.

In the second step, the partially silylated dextran was used as a multifunctional macroinitiator for the ROP of D,L-lactide in the presence of a low amount of tin activator (SnOct2).23 All the results obtained by NMR as well as SEC have demonstrated both the efficiency of the PLA grafting onto the silylated dextran backbone and the absence of significant transesterification. The deprotection was performed under very mild acidic conditions23 in the last step, leading to amphiphilic dex-g-PLA. Finally, one can see from Table 1 that synthesized copolymers have quite similar average number of grafts (y) and with very different graft lengths from 144 to 2300 g/mol. Thus average PLA weight fractions (FPLA) varied considerably from one copolymer to another from 0.15 up to around 0.83. The PLAgrafted dextran copolymers exhibited different solubility characteristics depending on the PLA/dextran ratio: those with high dextran/PLA ratios tend to be water soluble (Table 1, entries 2, 3, 4), whereas the one with high FPLA was soluble in organic solvents like toluene as well as more polar solvents like dichloromethane or glycerol triacetate (Table 1, entry 1). In previous papers,22,23 we rapidly mentioned that these copolymers were able to adapt their conformation in order to be soluble in various solvents. A core–shell conformation whose structure depends on the solubility of copolymers in the studied solvents was thus proposed. 3.1.2. Synthesis of Hydrophobically Modified Dextrans DexC6y. The synthesis of dextran derivatives by random attachment of hydrocarbon groups through the formation of ether bonds was already reported in detail, and the dextran derivatives used in that work were characterized.15 Their chemical structure is given on figure 1. These dextran derivatives will be named DexC6y where y is the average number of hydrocarbon tails grafted within 100 glucose units. Consequently, the meaning of y is the same as for Dex-g-PLA except that PLA grafts are replaced by hydroxylated octyl groups. 3.1.3. Surface ActiVe Properties of Amphiphilic Dextran DeriVatiVes. The surface active properties of PLA-grafted dextran copolymers as well as those of hydrophobically modified dextrans have been characterized previously either at air-water or oil-water interfaces. For a detailed discussion of the links between the chemical structure and the surface active behavior of each polymer series, the reader is referred to previous papers.8,16,22 Nevertheless, we will try to compare rapidly the equilibrium surface active properties of both families of dextran derivatives, Dexn-g-yPLAm and DexC6y at the air-water interface (Table 2). Only the equilibrium values will be briefly discussed here; the kinetics of surface tension decrease are not considered. For comparison, the values reported for a polymeric surfactant derived from inulin (polyfructose) are reported. Surface tension levels down with increasing polymer concentration and reaches the maximum surface pressure (ΠC) above a



Table 2. Surface Active Properties of Various Neutral Amphiphilic Polysaccharides at the Air–Water Interface polymera

ΠC (mN/m)

CC (g/L)

Γmax (mol/m2)

minimal weight excess (mg/m2)c

ref

DexC612 Dex29K-g-18PLA0.1K Dex29K-g-14PLA0.2K octyl modified inulinb

∼36 12 17 39

∼15 0.2 0.9 0.1

1.2 × 10-6 1.7 × 10-6 1.0 × 10-6 1.8 × 10-6

1.7 1.7 1.4 3.2

this work 22 22 7

a The mean number of hydrophobic groups (hydroxylated octyl groups, PLA grafts, or octyl groups) per one dextran chain is: 19 for DexC612, 32 for Dex29K-g-18PLA0.1K, 25 for Dex29K-g-14PLA0.2K, and 3 for octyl modified inulin. b This amphiphilic polysaccharide was prepared by chemical modification of a commercial sample of inulin (polyfructose) having a degree of polymerization around 25 by octyl chains with a degree of substitution of about 10%.7 c For the estimation of the minimal weight surface excess, see text.

critical concentration, noted CC. The maximum surface excess, Γmax, deduced directly from surface tension measurements using the Gibbs equation, is expressed in moles of adsorbing groups per units of area. In the case of amphiphilic polymers, adsorbed groups are the glucosidic units carrying hydrophobic tails. This maximum surface excess can be converted into a mass excess of adsorbed polymer if the mass fraction of the adsorbed groups is known. It is then necessary to assume that all the hydrophobic tails of adsorbed macromolecules are in direct contact with the interface (which means that they are included in the value of Γmax).44 In case this assumption was not correct, the real mass excess of adsorbed polymer would be higher, which means that, in that way, it is estimated by default. The maximum surface excess, Γmax, and the minimal weight surface excess are similar. The obtained values are much lower than those reported for nonionic saccharide surfactants (around 4 × 10-6 mol/m2) which is a consequence of the steric hindrance associated to polysaccharide coils. The surface pressure at the critical concentration is much lower for Dexn-g-yPLAm when compared to other hydrophobically modified derivatives of dextran or inulin. This is clearly related to the chemical nature of hydrophobic tails. Hydrocarbon groups are more hydrophobic than PLA side chains and lead to significantly higher surface pressure, ΠC. For hydrophobically modified derivatives of dextran or inulin, ΠC is between 35 and 40 mN/m, which is similar to that found with nonionic saccharide surfactants.45 3.2. Emulsifying Properties: Preparation of Direct and Inverse Miniemulsions. In what follows, emulsifying properties of PLA-grafted-dextran copolymers will be examined with the aim of obtaining submicrometric emulsions. According to Bancroft’s rule, direct emulsions should be obtained when a water-soluble surfactant is used, while inverse emulsions should result from the use of an oil-soluble surfactant.46 Even if this rule appeared to be invalid in some cases, it is still a convenient guideline for the design of polymeric surfactants. Graft copolymers with a low PLA fraction (FPLA) keep a convenient water solubility and will be studied as emulsifiers for direct emulsions. On the contrary, graft copolymers containing a significant FPLA (for instance, 50 wt %) can be solubilized in oils like dichloromethane or glycerol triacetate. These polymers will be considered as macromolecular surfactants for inverse emulsions. In what follows, the concentrations of solutes will always refer to the volume of the phase in which they are solubilized and not to the total volume of the emulsion. 3.2.1. Direct Emulsions. Indeed, amphiphilic copolymers carrying short PLA grafts appeared to be efficient as stabilizers in the preparation of submicrometric oil-in-water emulsions (Figure 2). Two different oils were used for those experiments: a nonpolar oil, dodecane, and a more polar oil, miglyol 810, which is a mixture of triglycerides.18 The average droplet diameter obtained immediately after the emulsification step could be lowered down to around 160 nm by changing either

Figure 2. Initial droplet diameter of oil-in-water emulsions as a function of the polymer-to-oil weight ratio. The oil is either dodecane (bold symbols) or mygliol 810 (open symbols). The polymer is either Dex29Kg-14PLA0.2K (circles) or DexC612 (squares).

polymer concentration in the aqueous phase or the oil volume fraction. The relevant parameter was shown to be the weight ratio of polymeric surfactant to oil as previously established.47 For a given amount of oil and polymer, the average droplet diameter was always lower with miglyol 810 than with dodecane. The same result was obtained with other hydrophobically modified dextrans and may be attributed to the lower interfacial tension between miglyol 810 and water (Table 3). For high polymer-to-oil ratios, the droplet diameter levels down to a limiting value that seems to be of the order of 150 nm for both oils. This value seems to be a characteristic of the emulsification process itself. These results can be compared to those obtained with a hydrophobically modified dextran DexC612. The same trends are observed (variation of droplet size with polymer-to-oil ratio, effect of oil nature) with the exception that the droplet diameters are generally lower with DexC612, everything else being equal. Nevertheless, at high polymer-to-oil ratios, droplet size levels down to about 150 nm, a similar value to that observed with Dex29K-g-14PLA0.2K. This result is consistent with the assumption that this minimum size should be related to the emulsifying process rather than to the polymeric surfactant itself. 3.2.2. InVerse Emulsions. A graft copolymer like Dex33K-gPLA 36 2.3K has a similar number of PLA side chains per dextran backbone as Dex33K-g-39PLA0.1K but PLA grafts are much longer. Consequently, Dex33K-g-36PLA2.3K exhibits a very low solubility in water and a significant solubility in glycerol triacetate. Glycerol triacetate (GTA) is widely used for the preparation of nanoparticles with biomedical applications. This oil was chosen for the preparation of inverse emulsions. The solubility of water-in-glycerol triacetate is rather high (Table 3). Consequently, submicrometric emulsions of water in GTA undergo very fast Ostwald ripening and cannot be kept during enough time for any measurement. The stability of such inverse


Raynaud et al.

Table 3. Physical Properties of Oils Used for Direct and Inverse Miniemulsionsa. oil decane dodecane miglyol 810 glycerol triacetate

Vm (m3/mol) -4

1.94 × 10 2.27 × 10-4 1.88 × 10-4

γiow (mN/m)

C∞oil (mol/m3) -4

52.3 52.8 22.5 3.3

C∞water (mol/m3)

D (m2/s) -10

3.6 × 10 2.4 × 10-5

0.366 0.283

5.9 × 10 5.4 × 10-10

3 × 102

1.9 103

2.0 × 10-9 b

refs 60,61 60,61 18 62–64

a

Vm, γiow, C∞oil, C∞water, D are the molar volume, the oil-water interfacial tension, the solubility of the oil in water, the solubility of water in the oil, and the diffusion coefficient in water, respectively (see eq 2). b Calculated using the correlation of Wilke and Chang.53

Figure 3. Initial droplet diameter of water-in-oil emulsions as a function of polymer concentration in the oil. The polymer used is Dex33K-g36PLA2.3K and the oil is glycerol triacetate. The salt dissolved in the aqueous phase is either NaCl (open symbols) or Na2SO4 (bold symbols). The water volume fraction is 9.1%, and the salt concentration in water is 0.5 mol/L.

Figure 4. Variation of average droplet size with time for oil-in-water emulsions prepared with Dex29K-g-14PLA0.2K at a concentration of 5 g/L in the aqueous phase. The oil volume fraction is 5% and the oil used is either decane (() or dodecane (b). The curve fittings were obtained with eq 1.

emulsions may be lengthened by adding hydrophilic solutes to the aqueous phase: glucose or salts are common examples.48,49 Water-in-oil miniemulsions were prepared in the presence of Dex33K-g-36PLA2.3K, previously dissolved in GTA. For these experiments, two different salts were added in the aqueous phase: sodium chloride and sodium sulfate, both at a concentration of 0.5 M. Sodium sulfate led to the lower initial droplet sizes, especially when the polymer concentration was low (Figure 3). This effect of salt nature will be discussed later, in relation to emulsion aging (see Section 3.3.3). Varying the concentration of polymeric surfactant in the oil phase allowed the preparation of inverse miniemulsions with an initial average droplet diameter of around 170 nm. 3.3. Stability of Miniemulsions. 3.3.1. General Characteristics of Ostwald Ripening (or Molecular Diffusion). Emulsions are thermodynamically unstable systems (except microemulsions). Various mechanisms are involved in emulsion aging. Miniemulsions stabilized by hydrophobically modified dextrans were shown to undergo aging exclusively through Ostwald ripening (also called molecular diffusion), at least within the first weeks following their preparation.11,14 Three main characteristics of this aging mechanism have been evidenced with these miniemulsions. The distribution of droplet size is entirely displaced up to higher diameters with a complete disappearance of the smallest droplets.11 The cube of the number-average droplet radius varies linearly with time, the slope being usually called the “rate of Ostwald ripening” (eqs 1and 2). Finally, the aging rate varies significantly with the solubility of the oil in water.

tension (N/m), D and C∞ are the diffusion coefficient (m2/s) and the solubility (mol/m3) of the dispersed phase in the continuous one, respectively, Vm is the molar volume of the dispersed phase (m3/mol), R is the gas constant (8.314 J/mol K), and T is the absolute temperature (K). Following the suggestion of Taylor,50 the raw values of ω deduced from photocorrelation spectroscopy measurements will be divided by 1.48 in order to take into account the difference between the intensity-averaged droplet radius (experimental data) and the number-averaged droplet radius (which appears in eq 1). 3.3.2. Aging of Direct Miniemulsions. The aging of direct emulsions stabilized by Dex29K-g-14PLA0.2K is conveniently depicted by eq 1 when hydrocarbon oils are used (Figure 4). It has been shown that emulsion aging by coalescence can be depicted by an equation formally similar to eq 1. Nevertheless, these results were obtained with oil-in-water emulsions with polymeric stabilizers dissolved in the oil phase and having a very low affinity for the aqueous phase. In that case, the steric stabilization of droplets against coalescence was almost inexistent. In this work, the amphiphilic polymers are soluble in the aqueous phase, with dextran sequences providing an efficient steric barrier. Consequently, we can reasonably assume that coalescence is not involved in emulsion aging. Furthermore, when comparing miniemulsions of dodecane and decane prepared in the same conditions, the aging is much faster for decane than for dodecane, which is also consistent with the Ostwald ripening mechanism because the solubility of decane in water is 15 times higher than that of dodecane (Table 3, Figure 4). Assuming that the interfacial tension is unmodified when changing decane to dodecane at a given polymer concentration, we can calculate the ratio of the aging rates of the respective emulsions to be around 12. The experimental values found with Dex29K-g-14PLA0.2K and DexC612 are 17 and 15, respectively, in rather good agreement with the previous estimation based on eq 2.

R3(t) ) R3(0) + ωt ω)

8γi DVm2 C∞ 9RT

(1) (2)

In eq 1, R is the number-average droplet radius (m) and ω is the “rate of Ostwald ripening” (m3/s). In eq 2, γi is the interfacial


Figure 5. Aging rate of direct and inverse emulsions as a function of polymer-to-dispersed phase weight ratio. Dodecane-in-water emulsions stabilized by Dex29K-g-14PLA0.2K (b) or DexC612 (O) or Na2SO4 0.5 M-in-glycerol triacetate emulsions stabilized by Dex33K-g-36PLA2.3K (().

The aging rates determined with Dex29K-g-14PLA0.2K can be compared to those obtained for miniemulsions prepared in the presence of a hydrophobically modified dextran, DexC612 (Figure 5). For dodecane emulsions containing relatively low amounts of polymer (polymer-to-oil weight ratio lower than 0.1), the aging rate is higher with Dex29K-g-14PLA0.2K than with DexC612. For higher amount of polymer, the aging rates seem to become similar, at least in the investigated range. This result is consistent with previous ones regarding the droplet size immediately after emulsification and could be related to the surface active properties of the polymers. Indeed, the lower the polymer-to-oil weight ratio, the remaining polymer concentration in the aqueous phase is very low, which leads to higher interfacial tensions. Furthermore, the Dex29K-g-14PLA0.2K copolymer being less surface active than the DexC612 gives the highest aging rates at low polymer-to-oil weight ratios (Figure 5). 3.3.3. Aging of InVerse Miniemulsions. The oil used for the continuous phase was GTA and the dispersed phase was water containing a solute at a concentration of 0.5M. Four different solutes were used: glucose, sodium chloride, sodium carbonate, and sodium sulfate. When comparing the aging process of four inverse miniemulsions differing by the nature of the solute present in the aqueous droplets, the aging rate varies in the following order: glucose > sodium chloride > sodium carbonate > sodium sulfate (Figure 6). The rise of droplet size in the case of glucose was too sharp for being plotted in Figure 6. This strong influence of the nature of the solute dissolved in the dispersed phase can be explained assuming that Ostwald ripening is the main aging process of inverse miniemulsions, at least during the first week following their preparation. Changing the solute present in the aqueous phase of an inverse emulsion is analogous to changing the nature of the oil in a direct emulsion because the nature of the salt has an effect on the shape of the ternary diagram (water, salt, GTA) and hence on the equilibrium amount of water in GTA. For instance, some salts have been reported to produce complete miscibility in their ternary mixtures with water and GTA.51 On the contrary, sodium chloride has been reported to reduce the amount of water in the organic phase.52 In addition, the solubility of salt being very low in the organic phase, the diffusion of water out of droplets is counterbalanced by the osmotic pressure in the droplets. This second effect is certainly predominant in the observed results about emulsion aging.


Figure 6. Droplet size as a function of time for water-in-glycerol triacetate emulsions with different salts dissolved in the aqueous phase: sodium chloride (O), sodium carbonate (9), and sodium sulfate (b). The polymer used was Dex33K-g-36PLA2.3K at a concentration of 5 g/L in the oil, and the water volume fraction is 9.1%. The lines are guides for the eye.

Miniemulsions containing sodium sulfate in the aqueous dispersed phase undergo the slower aging (Figure 6). Changing the salt from sodium chloride to sodium sulfate decreases the aging rate of approximately 2 orders of magnitude, down to 2.6 × 10-26 m3/s at a polymer concentration of 5 g/L. The aging rate of a water-in-GTA miniemulsion containing no surfactant should be around 2.5 × 10-22 m3/s, using the values of Table 3 and the following physical properties: Vm ) 1.805 × 10-5 m3/mol for water, D ) 3.4 × 10-10 m2/s for water in GTA (estimated by the correlation of ref 53). The values of aging rates can be converted into time scales, calculating the time needed for doubling the initial droplet size. For an emulsion of water in GTA, this time would be around 4 min. On the contrary, for an emulsion of Na2SO4 0.5 M in GTA with a Dex-g-PLA surfactant, this time is of the order of 500 h! The effect of solute nature on aging rate is consistent with the variation of the initial droplet size (Figure 3). We can assume that, during the emulsification, the polymer adsorption onto droplet surface must compete with two phenomena, leading to an increase of the droplet size: coalescence and Ostwald ripening. After the emulsification step, the adsorbed layer of macromolecules is dense enough to prevent coalescence so that Ostwald ripening may be the main aging process. The increase of the average droplet size of inverse emulsions with time is rather consistent with eq 1 (Figure 7), additional evidence of Ostwald ripening phenomenon. We must notice that, for the longest aging times, the droplet size seems to stop increasing, which could be attributed to the effect of the salt dissolved in water. The value of the aging rate was deduced from the first part of the plot as indicated in the inset of Figure 7. Actually, higher polymer-to-aqueous phase weight ratios decrease the aging rate down to a minimal value (Figure 5). A similar variation has been observed with direct miniemulsions either with graft copolymers or with hydrophobically modified dextrans using the weight ratio of polymeric surfactant to dispersed phase (see Section 3.3.2). 3.4. Nanoparticles of Polyacrylamide Networks by Inverse Miniemulsion Polymerization. A covalent hydrogel is a three-dimensional network of hydrophilic polymers that swells in aqueous solutions. Hydrogels have been widely explored for their applications in drug delivery vehicules,54 sensors, biological laboratory devices,55 separation and purification processes,56 recognition of biomolecules and proteins,57 or


Figure 7. Variation of average droplet size with time for water-inglycerol triacetate emulsions prepared with Dex33K-g-36PLA2.3K at a concentration of 5 g/L (b) or 7 g/L (O) in the oil. The aqueous phase contains Na2SO4 at a concentration of 0.5 mol/L, and its volume fraction is 9.1%. The curve fittings were obtained with eq 1.

tissue engineering.58 Hydrogel micro- and nanoparticles are interesting candidates for drug delivery applications.34 They are mechanically more stable than liposomes and exhibit a greater loading capacity. Furthermore, most of them are sensitive to external stimuli such as pH, temperature, or ionic strength.35 In this work, we focused on polyacrylamide (PAAm) nanoparticles. Such nanoparticles were studied because of the pH sensitivity of PAAm gels.36 Applications of such sensitive polymers as drug delivery systems39,40 or oxygen carriers have been already mentioned.41,42 There is a great interest for the preparation of hydrogels under the form of nanoparticles. Because of their high surface-to-volume ratio, such particles undergo fast swelling-deswelling transitions. Inverse miniemulsion polymerization is a convenient method to prepare hydrophilic nanoparticles.27 It was shown a few years ago that the principles of direct miniemulsion polymerization hold for inverse systems. Aqueous solutions of hydrophilic monomers could be dispersed in nonpolar solvents such as cyclohexane or hexadecane. Salts are used as lipophobe to reduce Ostwald ripening, while amphiphilic copolymers are mostly used to stabilize the droplets during the polymerization process. Although miniemulsion polymerization is now a rather well-documented process, only a few studies involve polymeric stabilizers.28–33 The use of polymeric stabilizers in a miniemulsion process combines the advantages of the surface coverage by macromolecules and the submicrometric dispersion of the reaction medium. To the best of our knowledge, the use of a biocompatible and degradable copolymer based on polysaccharide as stabilizer of inverse systems was never reported. Biocompatible amphiphilic polymers as stabilizers and triglyceride oil as the continuous phase are attractive components for the elaboration of network nanoparticles, which could find applications in the biomedical field. An aqueous solution of acrylamide, bisacrylamide, sodium sulfate was dispersed in glycerol triacetate containing dissolved Dex33K-g-36PLA2.3K. Sodium sulfate was used as a lipophobe on the basis of previous results (see Section 3.3.3). Bisacrylamide (3 wt % of the acrylamide) allows the cross-linking of the hydrogel. Inverse miniemulsion polymerization was performed at 75 °C for 2 h. Indeed, previous studies showed that the kinetics of the inverse miniemulsion polymerization of acrylamide is very fast.59 Droplet and particle sizes strongly depend on the concentration of the amphiphilic copolymer Dex33K-g-36PLA2.3K in the continuous phase (Figure 8). For concentrations above 2.5g/L,

Raynaud et al.

Figure 8. Droplet and particle size as a function of Dex33K-g-36PLA2.3K concentration in glycerol triacetate for acrylamide miniemulsion polymerization. (O) Droplet size, (b) particle size after polymerization of the monomer.

particle size was close to the initial droplet size and the coagulate amount was below 5 wt %. Thus, in those conditions, the particle size can be controlled by the initial emulsion characteristics. However, at lower polymer concentrations, particle size was much higher than the initial droplet size, indicating a partial destabilization of the miniemulsion during the polymerization step. A minimal concentration of 2.5 g/L is thus required to prevent particle aggregation. The scanning electron microscopy pictures of the obtained nanoparticles proved that the size distribution is relatively narrow despite a few bigger particles. These results are directly related to the macromolecular structure of the stabilizers. In inverse miniemulsion, the polysaccharide backbone is expected to be adsorbed at the interface, close to the aqueous phase, while the PLA grafts protrude into the continuous oil phase. Consequently, the steric protection of aqueous droplets is brought by the PLA side chains. The graft copolymer employed has rather short PLA grafts (six lactide units per graft on average) so that its ability to confer steric protection is certainly limited. As a result, a minimum amount of graft copolymer is required to avoid a significant size increase during polymerization. According to our opinion, macromolecular engineering might significantly improve the performance of the inverse miniemulsion process through the properties of the polymeric stabilizers.

4. Conclusion PLA-grafted dextran copolymers were synthesized and examined as stabilizers for submicrometric direct and inverse miniemulsions. The controlled synthesis allowed the preparation of copolymers with given lengths and numbers of PLA grafts. According to their structural characteristics, the graft copolymers were mainly soluble either in water or in organic solvents (glycerol triacetate or dichloromethane saturated with water). These copolymers exhibited efficient properties of stabilization of submicrometric direct or inverse miniemulsions depending on their chemical structure. The emulsions characteristics and stability were analyzed and appeared to be similar to those obtained with other hydrophobically modified dextrans. Inverse miniemulsion polymerization was carried out in the presence of such Dex-g-PLA stabilizers and allowed the preparation of hydrogel nanoparticles with controlled characteristics.


The two synthetic strategies mentioned in that work (chemical modification of polysaccharide by the attachment of hydrocarbon groups and “grafting from” polymerization of low-polarity side chains) both allow the control of emulsifying properties of the resulting polymeric surfactants. By the chemical modification pathway, the lipophilicity of the modified polysaccharide can be tuned by choosing the hydrocarbon group and varying the number of attached groups per chain. The “grafting from” polymerization allows adjusting the lipophilicity of the copolymer by changing the number of side chains as well as their average length. The biodegradable amphiphilic polymers studied here exhibit efficient properties as stabilizers of submicrometric dispersions containing biocompatible oils like triglycerides. These dispersions can serve for the elaboration of drug delivery systems with controlled characteristics (size, surface coverage, chemical structure, . . .). More work is currently being carried out in order to document the relations between the chemical structure and the emulsifying properties as well as to improve the stabilization during miniemulsion polymerization.

References and Notes (1) Landoll, L. M. J. Polym. Sci., Polym. Chem. Ed. 1982, 20, 443–455. (2) Wollenweber, C.; Makievski, A. V.; Miller, R.; Daniels, R. J. Colloid Interface Sci. 2000, 117, 419–426. (3) Akiyoshi, K.; Deguchi, S.; Tajima, H.; Nishikawa, T.; Sunamoto, J. Macromolecules 1997, 30, 857–861. (4) Babak, V. G.; Lukina, I. G.; Vikhoreva, G. A.; Desbrières, J.; Rinaudo, M. Colloids Surf., A 1999, 147, 139. (5) Babak, V. G.; Skotnikova, E. A.; Lukina, I. G.; Pelletier, S.; Hubert, P.; Dellacherie, E. J. Colloid Interface Sci. 2000, 225, 505. (6) Grant, S.; Blair, H. S.; McKay, G. Polym. Commun. 1990, 31, 267– 268. (7) Stevens, C. V.; Meriggi, A.; Peristeropoulou, M.; Christov, P. P.; Booten, K.; Levecke, B.; Vandamme, A.; Pittevils, N.; Tadros, T. F. Biomacromolecules 2001, 2, 1256. (8) Rouzes, C.; Durand, A.; Léonard, M.; Dellacherie, E. J. Colloid Interface Sci. 2002, 253, 217–223. (9) Fournier, C.; Léonard, M.; Le Coq-Leonard, I.; Dellacherie, E. Langmuir 1995, 11, 2344–2347. (10) Rouzes, C.; Gref, R.; Léonard, M.; Delgado, A. D.; Dellacherie, E. J. Biomed. Mater. Res. 2000, 50 (4), 557–565. (11) Sadtler, V. M.; Imbert, P.; Dellacherie, E. J. Colloid Interface Sci. 2002, 254, 355–361. (12) Rouzes, C.; Leonard, M.; Durand, A.; Dellacherie, E. Colloids Surf., B 2003, 32, 125–135. (13) Fournier, C.; Leonard, M.; Dellacherie, E.; Chikhi, M.; Hommel, H.; Legrand, A. P. J. Colloid Interface Sci. 1998, 198, 27–33. (14) Rotureau, E.; Leonard, M.; Dellacherie, E.; Durand, A. Phys. Chem. Chem. Phys. 2004, 6, 1430–1438. (15) Rotureau, E.; Chassenieux, C.; Dellacherie, E.; Durand, A. Macromol. Chem. Phys. 2005, 206, 2038–2046. (16) Rotureau, E.; Leonard, M.; Dellacherie, E.; Durand, A. J. Colloid Interface Sci. 2004, 279, 68–77. (17) Aumelas, A.; Serrero, A.; Durand, A.; Dellacherie, E.; Léonard, M. Colloids Surf., B 2007, 59, 74–80. (18) Mosqueira, V. C. F.; Legrand, P.; Pinto-Alphandary, H.; Puisieux, F.; Barratt, G. J. Pharm. Sci. 2000, 89, 614–626. (19) de Jong, S. J.; de Smedt; S. C.; Demeester, J.; van Nostrum, C. F.; Kettenes-van den Bosch, J. J.; Hennink, W. E. J. Controlled Release 2001, 72, 47–56. (20) Gref, R.; Rodrigues, J.; Couvreur, P. Macromolecules 2002, 35, 9861– 9867. (21) Ouchi, T.; Saito, T.; Kontani, T.; Ohya, Y. Macromol. Biosci. 2004, 4, 458–463. (22) Nouvel, C.; Frochot, C.; Sadtler, V.; Dubois, P.; Dellacherie, E.; Six, J.-L. Macromolecules 2004, 37, 4981–4988.

Biomacromolecules, Vol. 9, No. 3, 2008 1021 (23) Nouvel, C.; Ydens, I.; Degée, P.; Dubois, P.; Dellacherie, E.; Six, J.-L. J. Polym. Sci., Part A: Polym. Chem. 2004, 42, 2577. (24) Nouvel, C.; Dubois, P.; Dellacherie, E.; Six, J.-L. Biomacromolecules 2003, 4, 1443–1450. (25) Nouvel, C.; Ydens, I.; Degée, P.; Dubois, P.; Dellacherie, E.; Six, J.-L. Polymer 2002, 43, 1735–1743. (26) Thanki, P. N.; Dellacherie, E.; Six, J.-L. Eur. Polym. J. 2005, 41, 1546–1553. (27) Landfester, K.; Willert, M.; Antonietti, M. Macromolecules 2000, 33, 2370–2376. (28) Ou, J.-L.; Lim, M. S.; Chen, H. J. Appl. Polym. Sci. 2003, 87, 2230. (29) Houillot, L.; Nicolas, J.; Save, M.; Charleux, B.; Li, Y.; Armes, S. P. Langmuir 2005, 21, 6726. (30) Lim, M. S.; Chen, H. J. Polym. Sci., Part A: Polym. Chem. 2000, 38, 1818. (31) Baskar, G.; Landfester, K.; Antonietti, M. Macromolecules 2000, 33, 9228. (32) Marie, E.; Landfester, K.; Antonietti, M. Biomacromolecules 2002, 3, 475. (33) Ni, P.; Zhang, M.; Ma, L.; Fu, S. Langmuir 2006, 22, 6016. (34) Sahiner, N.; Singh, M. Polymer 2007, 48, 2827–2834. (35) Pich, A.; Tessier, A.; Boyko, V.; Lu, Y.; Adler, H.-J. P. Macromolecules 2006, 39, 7701–7707. (36) Qiu, Y.; Park, K. AdV. Drug DeliVery ReV. 2001, 53, 321–329. (37) Guilherme, M. R.; Reis, A. V.; Paulino, A. T.; Fajardo, A. R.; C.; Muniz, E.; Tambourgi, E. B. J. Appl. Polym. Sci. 2007, 105, 2903– 2909. (38) Ni, H.; Kawaguchi, H.; Endo, T. Colloid Polym. Sci. 2007, 285, 873– 879. (39) Jain, S.; Yap, W. T.; Irvine, D. J. Biomacromolecules 2005, 6, 2590– 2600. (40) Agnihotri, S. A.; Aminabhavi, T. M. Int. J. Pharm. 2006, 324, 103– 115. (41) Patton, J. N.; Palmer, A. F. Biomacromolecules 2005, 6, 414–424. (42) Patton, J. N.; Palmer, A. F. Langmuir 2006, 22, 2212–2221. (43) Ydens, I.; Rutot, D.; Degée, P.; Six, J.-L.; Dellacherie, E.; Dubois, P. Macromolecules 2000, 33, 6713–6721. (44) Millet, F.; Nedyalkov, M.; Renard, B.; Perrin, P.; Lafuma, F.; Benattar, J.-J. Langmuir 1999, 15, 2112–2119. (45) Zhang, T.; Marchant, R. E. J. Colloid Interface Sci. 1996, 177, 419– 426. (46) Bancroft, W. D. J. Phys. Chem. 1913, 17, 501–519. (47) Rotureau, E.; Leonard, M.; Marie, E.; Dellacherie, E.; Camesano, T. A.; Durand, A. Colloids Surf., A 2006, 288, 131–137. (48) Kizling, J.; Kronberg, B. Colloids Surf. 1990, 50, 131–140. (49) Koroleva, M. Y.; Yurtov, E. V. Colloid J. 2003, 65, 40–43. (50) Taylor, P. AdV. Colloid Interface Sci. 1998, 75, 107. (51) Raridon, R. J.; Kraus, K. A. J. Colloid Sci. 1965, 20, 1000–1013. (52) Kraus, K. A.; Raridon, R. J.; Baldwin, W. H. J. Am. Chem. Soc. 1964, 86, 2571–2576. (53) Wilke, C. R.; Chang, P. AIChE J. 1955, 1, 264. (54) Delgado, M.; Spanka, C.; Kerwin, L. D.; Wentworth, P.; Janda, K. D. Biomacromolecules 2002, 3, 262–271. (55) Martin, B. D.; Gaber, B. P.; Patterson, C. H.; Turner, D. C. Langmuir 1998, 14, 3971–3975. (56) Cai, W.; Gupta, R. B. Ind. Eng. Chem. Res. 2001, 40, 3406–3412. (57) Sergeyev, V. G.; Novoskoltseva, O. A.; Pyshkina, O. A.; Zinchenko, A. A.; Rogacheva, V. B.; Zezin, A. B.; Yoshikawa, K.; Kabanov, V. A. J. Am. Chem. Soc. 2002, 124, 11324–11333. (58) Miralles-Houzelle, M. C.; Hubert, P.; Dellacherie, E. Langmuir 2001, 17, 1384–1391. (59) Capek, I. Des. Monomers Polym. 2003, 6, 399–409. (60) Sakai, T.; Kamogawa, K.; Nishiyama, K.; Sakai, H.; Abe, M. Langmuir 2002, 18, 1985. (61) Jiao, J.; Burgess, D. J. J. Colloid Interface Sci. 2003, 264, 509–516. (62) Benerito, R. R.; Singleton, W. S.; Feuge, R. O. J. Phys. Chem. 1954, 58, 831–834. (63) Hefter, G. T. Solubility Data Ser. 1992, 49, 134–137. (64) Rodriguez, M.; Galan, M.; Munoz, M. J.; Martin, R. J. Chem. Eng. Data 1994, 39, 102–105.

BM701101N

Emulsifying Properties Of Biodegradable Polylactide-Grafted Dextran

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