Physicochemical evaluation of PLA nanoparticles stabilized by water-soluble MPEO–PLA block copolymers

Journal of Colloid and Interface Science 268 (2003) 441–447 www.elsevier.com/locate/jcis

Physicochemical evaluation of PLA nanoparticles stabilized by water-soluble MPEO–PLA block copolymers D. Chognot,a J.L. Six,a M. Leonard,a,∗ F. Bonneaux,b C. Vigneron,b and E. Dellacherie a a Laboratoire de Chimie Physique Macromoléculaire, UMR CNRS-INPL 7568, Groupe ENSIC, BP 451, 54001 Nancy Cedex, France b Laboratoire d’Hématologie–Physiologie, Faculté de Pharmacie, 5, rue Albert Lebrun, 54000 Nancy Cedex, France

Received 12 February 2003; accepted 28 May 2003

Abstract Different water-soluble MPEO–PLA diblock copolymers with various α-methoxy-ω-hydroxyl polyethylene (MPEO) and poly(lactic acid) (PLA) block lengths have been synthesized. Their surface-active properties were evidenced by surface tension (water/air) measurements. In each case the surface tension leveled down above a critical polymer concentration, which was attributed to the formation of a dense polymer layer at the liquid–air interface. The applicability of copolymers as emulsion stabilizers in the preparation of PLA nanospheres by an o/w emulsion/evaporation technique was then investigated. Four copolymers presenting sufficient water solubility and good surfactive properties were used to prepare PLA nanospheres with MPEO chains firmly anchored at the particle surface. The effect of polymer concentration in emulsion on particle size and surface coverage was examined. Whatever the copolymer characteristics, it was found that the optimal concentration to obtain a large amount of MPEO at the particle surface was similar (around 2 g/l). The effect of the copolymer composition on MPEO layer characteristics and on colloidal stability was also evaluated. The conformation of MPEO blocks at the PLA particle surface is discussed in relation to the layer thickness and the surface area occupied per molecule.  2003 Elsevier Inc. All rights reserved. Keywords: PLA particles; MPEO–PLA copolymers; Surface properties

1. Introduction Polymeric particles made of poly(lactic acid) (PLA) or its copolymer with glycolic acid with controlled surface properties have been increasingly studied for therapeutic applications such as controlled release and drug targeting. In particular, efforts to prevent rapid adsorption of plasma proteins and phagocyte activation have centered on modifying the particle surface with a hydrophilic polymer layer. For instance, it is well known that surface modifications with hydrophilic and neutral polymers such as poly(ethylene oxide) (PEO) or polysaccharides may reduce nonspecific protein adsorption and thus improve the biocompatibility of materials in contact with biological fluids [1–3]. The ability of surface-bound PEO or naturally occurring polysaccharides to resist protein adsorption has attracted considerable attention in the past two decades. Extensive reviews on the adsorption of biomolecules onto PEO-grafted * Corresponding author.

E-mail address: [email protected] (M. Leonard). 0021-9797/$ – see front matter  2003 Elsevier Inc. All rights reserved. doi:10.1016/S0021-9797(03)00591-5

surfaces have been reported which deal with the advantages of PEO chains in terms of immunogenicity, antigenicity, flexibility, and protein-repelling properties [4–7]. PLA particles are classically produced by emulsion– evaporation, emulsion–diffusion, or solvent displacement methods. These techniques are similar in that they involve an organic solution containing the drug and an aqueous solution containing the stabilizer. Polyvinyl alcohol (PVA) is commonly used as a colloidal polymeric stabilizer and provides particles with a hydrophilic polymer layer but has the disadvantage of not being accepted for intravenous administration. To overcome this problem, PVA can be replaced by more biocompatible polymers. Among them, PEO-based copolymers are of considerable interest because of their extremely low toxicity and low immunogenic response. For example, the use of PEO–PLA diblock copolymers in the organic phase of emulsion during particle formation provides a hydrophilic layer of PEO chains extending from the surface and anchored in the hydrophobic PLA shell by their PLA blocks [8]. However, the production of such particles— generally composed of PEO–PLA copolymer with long PLA

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chains and short PEO chains—by an emulsion process requires the use of a surfactant in the aqueous phase of the emulsion, whose subsequent removal from the particle surface cannot be easily performed [8–10]. The role of PEO-based polymers with different compositions as aqueous surfactants (e.g., PEO–PLA with short PLA chains [11] and, above all, poloxamers or poloxamine [12,13]) was also frequently reported over the past decade. The present paper deals with the preparation of watersoluble amphiphatic MPEO–PLA copolymers and their use as stabilizers in the o/w emulsion technique. The effect of various structural parameters, such as MPEO/PLA ratio and block lengths of MPEO and PLA, on the copolymer surfactive properties was investigated. The physicochemical properties of the nanospheres prepared in the presence of these copolymers were also studied, in terms of size, electrophoretic mobility, adsorbed layer thickness, surface density of MPEO, and stability of dispersions in the presence of salts.

2. Experimental 2.1. Materials n 20,000 g/mol), MPEO10K (M n 10,000 MPEO20K (M  g/mol) and MPEO5K (Mn 5000 g/mol) were purchased from Polymer Source (Lajoie, Canada). Toluene from Prolabo (Fontenay-Sous-Bois, France) was dried by refluxing it over CaH2 (for 12 h) and stored under nitrogen. Just before use, toluene was further dried over polystyryl lithium and freeze-distilled. D, L-lactide from Lancaster (Bischheim, Strasbourg, France) was twice purified by recrystallization from dry toluene and dried under reduced pressure. After dilution with dry toluene, 10−2 mol/l tin(II) 2-ethylhexanoate (Aldrich, St. Quentin Fallavier, France) solution was stored under nitrogen and used without any further purification. n = 45,000 g/mol, M w = Poly(D, L-lactide) (PLA45K: M 55,000 g/mol) and ethyl acetate were purchased from Sigma (St. Quentin Fallavier, France). Iodine and potassium iodide were from Prolabo and deionized water was used in all experiments. 2.2. Synthesis of the MPEO–PLA copolymers Nine MPEO–PLA copolymers with different MPEO/PLA ratios were synthesized by pseudo-anionic ring-opening polymerization of (D, L-lactide) from the hydroxyl end group of MPEO after activation with stannous octoate [14]. Typically, 10 g of MPEO were dried by three azeotropic distillations of toluene. After drying under reduced pressure for 24 h, a defined volume of toluene was added under nitrogen and the solution was kept at 50 ◦ C for 1 h. The resulting solution was added to defined amounts of purified D, L-lactide and the temperature was increased up to 100 ◦ C. Then a known amount of stannous octoate solution (molar

ratio [SnOct2]/[OH] = 0.03) was added and the polymerization was carried out at 100 ◦ C for 4 h. Deactivation was realized by addition of some drops of acidic methanol. The copolymer was recovered by two precipitations in cold ethyl ether and dried under vacuum. 2.3. Characterization of MPEO–PLA copolymers w of MPEO n and M The average molecular weights M 1 and copolymers were obtained from H NMR in CDCl3 (Bruker AC 300 MHZ HR-MAS spectrophotometer, Wissembourg, France) and multiangle laser light scattering (MALLS, Wyatt, Mini Dawn, Santa Barbara, USA) coupled to size exclusion chromatography (SEC). The MPEO/PLA ratios were calculated from the areas at the resonance peaks of the PLA methine group (δ = 5.2 ppm) and MPEO methylene groups (δ = 3.65 ppm). SEC of MPEO was first performed in 0.1 M NaNO3 (0.7 ml/min, serial set of SB-806-HQ, SB-805-HQ, SB-804-HQ OHPack columns D 8 µm: 300 × 8 mm and SB-OH Pack guard column: 50 × 6 mm, Shodex, Showa Denko, Dusseldorf, Germany). A refractive index increment of 0.134 was used for molecular weight calculation. SEC of copolymers and MPEO was also performed in THF (0.7 ml/min, column PLgel mixed D 5 µm: 300 × 7.5 mm, guard column PLgel 5 µm: 50 × 7.5 mm, Polymer Laboratory, Marseille, France). The specific refractive index increments of copolymers were calculated according to 
dn/dC = WMPEO (dn/dC)MPEO 
+ WPLA (dn/dC)PLA , (1) where W is the weight fraction calculated from 1 H NMR of copolymers. dn/dC values of 0.068 and 0.054 were used respectively for MPEO and PLA. Surface tension measurements were carried out at 25 ◦ C with a K8 surface tensiometer (Krüss, Germany) using the Wilhelmy technique. All samples were equilibrated for a time sufficient to reach constant readings (5 min to 1 h). 2.4. Preparation of PLA nanospheres stabilized by MPEO–PLA The nanospheres were prepared by an o/w emulsion method as follows: 0.4 ml of a PLA45K solution (25 mg/ml) in ethyl acetate were added under vigorous stirring to 4 ml of an aqueous solution containing MPEO–PLA copolymer at various concentrations, from 0.05% to 1% w/v, and with sodium cholate (0.3% w/v) for bare PLA particles. The mixture was then sonicated (pulsed mode, 10 W, 1 min in an ice bath) using a Vibracell model 600 W (Sonics & Materials Inc., Danbury, USA). The solvent was evaporated under vacuum for 1 h. Finally, the obtained suspension was centrifuged (35,000 g, 20 min) and the collected nanospheres were resuspended in water, then centrifuged again in order to remove the nonadsorbed MPEO–PLA. This purification procedure was repeated two times.

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2.5. Characterization of PLA nanospheres stabilized by MPEO–PLA

The interfacial MPEO concentration (δMPEO ) and the average distance between two MPEO chains (s) were calculated from the amount of MPEO–PLA at the particle surface and the MPEO weight fraction in the copolymer. The colloidal stability of nanosphere dispersions in the presence of added electrolyte was assessed by turbidimetry. Typically, 100 µl of dispersions were added to 3 ml of NaCl (from 1 × 10−4 to 4 M). The samples were allowed to stand for 40 min at ambient temperature and their absorbance was measured over the range 450–700 nm at 50 nm intervals. The slope of the straight line log(optical density) versus log(wavelength) was taken as an indication of particle aggregation [18].

Nanosphere size distribution (mean diameter and polydispersity index) was determined by photon correlation spectroscopy (PCS) using a Malvern spectrometer 4600 (Malvern Instruments, UK) in 10−3 M NaCl at 30 ◦ C. The electrophoretic mobility of nanospheres was determined using a Malvern Zetasizer 4 (Mavern Instruments,UK) and was studied in NaCl as a function of ionic strength. The zeta potential (ζ ) was calculated from the electrophoretic mobility using the modified Booth equation [15]. This equation allows the calculation of zeta potential for any k and a values, where k −1 is the Debye length and a the radius of particles, whereas the classical Smoluchowsky and Hückel equations are applicable only under two limiting cases, ka > 100 and ka < 0.1, respectively. The electrokinetic layer thicknesses (δPZ ) were calculated from the zeta potential evolution versus k using the Eversole and Boardman equation [16]. The specific surface area (Sp ) of the nanospheres in m2 /g was calculated according to Sp =

3 , (aρ)

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3. Results and discussion The compositions and molecular weights of synthesized water-soluble MPEO–PLA copolymers are given in Table 1. No trace of PLA homopolymer was observed from sizeexclusion chromatography experiments.

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3.1. Interfacial behavior of MPEO–PLA copolymers in aqueous solution

where a is the nanospheres’ mean radius determined by PCS and ρ the nanospheres’ density (here ρPLA = 1.26 × 106 g/m3 ). The amount of MPEO–PLA at the particle surface (Γ ) was also directly determined by UV spectrophotometry at 500 nm using the I2 /KI assay (1% I2 , 2% KI w/v) [17] (direct method) after nanosphere degradation (5 mg) in 1 M NaOH (1 ml) for 5 h. The MPEO surface coverage (ΓMPEO ) of the nanospheres was calculated according to mMPEO , ΓMPEO = (3) (mNS Sp )

The effect of MPEO–PLA on the steady state surface tension of water was measured as a function of the polymer concentration. MPEO exhibits a small surface activity resulting from attractive interactions between the PEO segments and the water/air interface [6]. However, the surface activity of water-soluble MPEO–PLA copolymers is in all cases significantly higher. As examples, the experimental results of the surface tension (σ ) versus polymer concentration for MPEO20K –PLA1.7K and MPEO20K–PLA2.9K are presented in Fig. 1. The results correspond to a classic surfactant behavior. Water surface tension decreases linearly with the increasing logarithm of surfactant concentration and then levels off.

where mMPEO is the MPEO amount at the particle surface and mNS is the mass of degraded particles. Table 1 Composition and molecular weights of copolymers under investigation Copolymer MPEO20K –PLA5.6K MPEO20K –PLA2.9K MPEO20K –PLA1.7K MPEO10K –PLA3.3K MPEO10K –PLA1.7K MPEO10K –PLA1.1K MPEO5K –PLA1.5K MPEO5K –PLA0.9K MPEO5K –PLA0.5K

n MPEO a M (g/mol)

n MPEO b M (g/mol)

MPEO contentc (% weight)

n PLA d M (g/mol)

n MPEO−PLA b M (g/mol)

w MPEO−PLA b M (g/mol)

19,350 19,350 19,350 10,000 10,000 11,000 5000 5000 5000

18,500 18,500 18,500 10,000 10,000 10,000 5300 5000 5300

77.6 87.2 92 75 85.3 91 77.2 83.8 91.2

5600 2900 1700 3300 1700 1100 1500 900 500

24,450 22,560 20,730 12,520 12,340 12,260 8130 6350 6520

25,840 24,430 21,290 13,390 13,600 12,550 8670 6700 7100

a Determined by size exclusion chromatography coupled to a multiangle laser light scattering detector (SEC-MALLS) with an aqueous eluent (0.1 M NaNO3 ). b Determined by SEC-MALLS in THF. c Determined by 1 H NMR. d Determined from 1 H NMR data and M n MPEO values as determined in footnote a.

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Table 2 Characteristics of the different MPEO–PLA surfactants Copolymer MPEO20K –PLA5.6K MPEO20K –PLA2.9K MPEO20K –PLA1.7K MPEO10K –PLA3.3K MPEO10K –PLA1.7K MPEO10K –PLA1.1K MPEO5K –PLA1.5K MPEO5K –PLA0.9K MPEO5K –PLA0.5K

ΠCMC (mN/m)a

CMC (µmol/l)

Γex b (µmol MPEO–PLA/m2 )

Ac (nm2 /MPEO–PLA chain)

12.7 17.2 21.4 18.7 19.4 24.1 25.6 24.5 25.5

3 50 100 8 150 300 50 70 100

0.7 1.0 1.2 1.0 0.9 1.2 1.6 1.2 1.1

2.3 1.7 1.5 1.7 1.8 1.4 1.0 1.4 1.1

a Π CMC = σCMC − σ0 , where σCMC and σ0 stand for the values of surface tension of polymer solutions at the critical concentration and of pure water,

respectively. b Γ ex is the interfacial surface excess. c A is the surface area occupied by one MPEO–PLA chain at the water/air interface.

Fig. 1. Plots of surface tension (σ ) of aqueous polymer solutions for increasing concentrations of MPEO20K –PLA2.9K (1), MPEO20K –PLA1.7K (P), and MPEO20K (!).

However, in some cases, two break points were observed in the surface tension curves. These two break points are commonly seen with polymeric emulsifiers and often attributed to the molecular weight polydispersity. In particular, Chang et al. [19] observed that the higher the polymer hydrophobicity, the lower the polymer concentration values at the two break points. They proposed that the first break point is associated with aggregation of high-molecular-weight species and the second one with aggregation of low-molecularweight species. In our experiments, no correlation was found between the first break point concentrations and the MPEO–PLA characteristics. Another explanation may be the possible change in MPEO–PLA conformation at the interface. At low polymer concentrations, both the PLA and MPEO blocks spread on the air/water interface. When the polymer concentration increases, the MPEO blocks begin to desorb gradually, forming a brush, while the PLA blocks remain anchored at the surface. The lower inflection point was attributed to the CMC (critical micellar concentration). The CMC and the surface pressure decrease ΠCMC (ΠCMC = σCMC − σ0 , where σCMC and σ0 are the values of the surface tension of polymer solu-

Fig. 2. Dependence of the surface pressure (ΠCMC ) on the PLA molecular weight in the copolymers (Q) and the interfacial surface excess (Γex , !).

tions at the critical micellar concentration and of pure water, respectively) are given in Table 2. The interfacial surface excess (Γex ) and the area occupied by one copolymer chain (A) were calculated from the linear part of the curve when the slope was maximum, using the Gibbs equation, dσ = −2.303RTΓex d log C,

(4)

where T is the absolute temperature (K), R = 8.314 J mol−1 K−1 is the ideal gas constant, and C is the MPEO– PLA molar concentration (mol/l). For similar MPEO blocks, the CMC of MPEO–PLA surfactants decreases when the PLA block size increases. These results are consistent with the hydrophobic domain being the predominant driving force for micellization in aqueous solution [20]. However, the higher the PLA block length, the lower the ΠCMC value for the 10K and 20K MPEO species. For shorter MPEO block lengths (MPEO5K ), higher ΠCMC values were obtained and remained similar whatever the PLA block length within the studied range. In this last case, it should be observed that the PLA blocks are also rather short. In Fig. 2, the ΠCMC values obtained with all MPEO–PLA copolymers are plotted versus Γex and PLA molecular weight in the copolymers. Unexpectedly, ΠCMC

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values can be roughly linearly related to Γex , indicating that the tensioactive activity per copolymer chain remains similar, whatever the copolymer characteristics. One may also observe that the PLA block size is an important parameter since the ΠCMC values increase with the decrease in PLA block length. These results reveal that the surfactants with shorter PLA chains pack better at the liquid–air interface, within the polymer range studied. On the other hand, no extended correlation between ΠCMC or Γex values and MPEO block length or MPEO/PLA ratio was clearly evidenced. However, one can expect that the anchoring/stabilizing moiety ratio also plays a role in the polymer adsorption. These results may be explained by the fact that PLA blocks adopt a less extended conformation at the interface than MPEO blocks do and thus undergo steric repulsions.

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Fig. 3. Dependence of the diameter of PLA nanospheres on the MPEO–PLA concentration in water.

3.2. Characteristics of PLA particles obtained with water-soluble MPEO–PLA surfactants The use of MPEO5K –PLA0.5K as a stabilizer in the o/w emulsification process led to particles with large diameter and poor stability. The hydrophobic PLA block of the copolymer was presumably too small to provide a sufficient anchorage in the hydrophobic PLA45K core of particles. On the other hand, the copolymers with larger PLA blocks (e.g., MPEO20K –PLA5.6K or MPEO10K–PLA3.3K) did not present a sufficient range of solubility in water to allow the preparation of well-defined particles, in spite of the presence of a large water-soluble MPEO block. In the following study, the four copolymers (MPEO20K– PLA1.7K , MPEO10K –PLA1.1K, MPEO5K –PLA1.5K, and MPEO5K –PLA0.9K) presenting sufficient water solubility, good surfactant properties and varying in MPEO and PLA block length were used to prepare nanospheres. The size distribution of nanospheres was evaluated before and after the washing and centrifugation steps. No significant change was observed with PLA particles covered with MPEO–PLA copolymers. On the other hand, PLA particles prepared in the presence of sodium cholate showed a strong tendency to aggregate, probably because of the cholate desorption during the washing steps. In this last case, the suspension had to be redispersed by sonication and then filtered before the characterization. With all these copolymers, the shapes of the curves giving the particle diameter versus the copolymer concentration were similar and showed, as classically, that the particle size decreased with increasing the polymer concentration in the emulsion aqueous phase (Fig. 3). However, slightly larger particles were obtained with MPEO20K –PLA1.7K at similar copolymer concentrations. At concentrations of MPEO– PLA equal to or higher than 2 g/l, about 200-nm particles with a weak polydispersity index (0.1 to 0.2, indicative of a relatively narrow particle size distribution) were obtained. The electrophoretic mobility of PLA particles, bare and covered with MPEO–PLA copolymers, was measured in NaCl as a function of the ionic strength. The evolution of

Fig. 4. Evolution of the zeta potential (ζ ) of PLA45K particles, bare and covered with different MPEO–PLA according to the ionic force.

zeta potential (ζ ) versus ionic strength is shown in Fig. 4. In all curves, it can be observed that |ζ | decreases with increasing NaCl concentration. Whatever the ionic strength, the bare PLA nanoparticles have a large negative ζ potential. This was attributed to the presence of ionized carboxyl groups on the nanosphere surface [21]. The PLA nanoparticles produced with copolymers as stabilizers were expected to have negligible surface charge, since the carboxyl acid end groups of the PLA core were supposed to be masked by the MPEO segment of copolymers which must shift the shear plane away from the surface [22]. As expected, all the nanospheres prepared in the presence of copolymers exhibited |ζ | values lower than bare nanospheres did, whatever the NaCl concentration. This reduction of particle ζ potential is slightly more important with the copolymer possessing the MPEO highest molecular weight while the length of PLA chains has no marked effect. This was assigned to the formation of a thicker MPEO layer at the particle surface. The amounts of MPEO remaining on the nanoparticles (ΓMPEO ) after solvent evaporation and subsequent washings, as well as the coating layer thickness (δPZ ), were determined as a function of the copolymer concentration in the emulsion water phase. The results obtained with MPEO20K–

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surface area occupied by one MPEO chain at the particle surface (A). s was estimated from the amount of adsorbed MPEO according to s = (M/ΓMPEO NA )1/2 ,

where M is the molecular weight of MPEO and NA the Avogadro number. The MPEO layer thickness determined from zeta potential measurement is compared with the equilibrium thickness L predicted by the Alexander–de Gennes expression as L = Ns −2/3 a 5/3 , where N is the MPEO polymerization degree, a the size of the PEO monomer unit, and s the average distance between two MPEO chains calculated according to Eq. (5) [23,24]. In this calculation, the monomer size was taken to be 0.4 nm [25]. Results are given in Table 4. For each copolymer, the thickness of the MPEO layer is of the same order as that predicted by the Alexander–de Gennes model and increases significantly with MPEO molecular weight. We previously showed that s ∼ Rg MPEO is the marking point at which MPEO chains start to extend away from polystyrene surfaces, resulting in chain stretching [26]. At present, the spacing between two MPEO chains is, in all cases, lower than or of the same order as the radius of gyration of MPEO (Rg MPEO ), meaning that the MPEO chains overlap and start to adopt a brushlike conformation. Rg MPEO was estimated as Rg MPEO = 0.181N 0.58 (nm), where N is the number of repeat units in the MPEO chain [27,28]. However, it should be noted that the surface area per molecule (A) is in the range 5–14 nm2 , that is, several times greater than that obtained from surface tension measurement. This means that less dense MPEO layers are formed at the PLA particle surface than at the water/air interface. This difference could be explained by the presence of PLA in the organic phase and/or by the PLA particle curvature that might reduce the available interfacial volume for anchoring the PLA block of the copolymer. Moreover, the MPEO block size appears to be a significant parameter for the characteristics of MPEO layer at the PLA particle surface since, at similar PLA block sizes, the surface area per molecule and the layer thickness increase with the MPEO molecular weight. The colloidal stability of MPEO–PLA particles was then examined at various NaCl concentrations. The differences in

Fig. 5. Dependence of surface coverage (ΓMPEO , P) and layer thickness (δPZ , ") on copolymer concentration in emulsion aqueous phase in the case of MPEO20K –PLA1.7K . Table 3 Characteristics of particles obtained with 2 g/l copolymer solutions Surfactant copolymer Noneb MPEO20K –PLA1.7K MPEO10K –PLA1.1K MPEO5K –PLA1.5K MPEO5K –PLA0.9K

Particle size (nm ± SD)

Polydispersity index

Zeta potentiala (mV)

155 ± 6.8 206 ± 6.9 195 ± 6.5 184 ± 4.4 194 ± 5.3

0.15 0.07 0.13 0.19 0.21

−45.0 −1.5 −2.4 −3.2 −3.4

(5)

a In 10−2 M NaCl. b Nanospheres were prepared in the presence of sodium cholate

(0.3% w/v).

PLA1.7K are reported in Fig. 5. At copolymer concentrations lower than 2 g/l, the higher the MPEO–PLA concentration, the higher both the amount of adsorbed copolymer and the layer thickness. At concentrations above 2 g/l, no further change was observed. With the other MPEO–PLA copolymers, plateau values were obtained at similar polymer concentration, i.e., 2 g/l. Characteristics of particles obtained with the four copolymers at this 2 g/l concentration are given in Table 3. Assuming that all the copolymer MPEO chains contribute to form the coating layer whereas the PLA bocks are anchored into the PLA45K core, it is then possible to calculate the average distance (s) between two MPEO chains and the Table 4 Characteristics of MPEO layers on particles Surfactant copolymer

ΓMPEO a (mg/m2 )

δPZ b (nm)

Ac (nm2 /MPEO chain)

s d (nm)

Le (nm)

Rg MPEO f (nm)

MPEO20K –PLA1.7K MPEO10K –PLA1.1K MPEO5K –PLA1.5K MPEO5K –PLA0.9K

3.31 1.33 1.52 1.64

32 17 13 12

11 14 6 5

3.3 3.7 2.4 2.3

45 20.8 13.8 14.2

6.3 4.5 2.8 2.8

a b c d e f

MPEO surface coverage. Electrokinetic layer thickness. Surface area occupied by one MPEO chain at the particle surface. Average distance between two MPEO chains. Equilibrium MPEO layer thickness predicted by Alexander–de Gennes [24]. Radius of gyration of a MPEO chain.

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flocculation provide a simple means to detect modification in the surface characteristics of PLA particles. Above 0.008 M NaCl, suspensions of bare PLA particles are destabilized, due to the screening of the surface charge by salt ions. Therefore flocculation due to van der Waals attraction occurs when increasing salt concentration. Whatever the copolymer we used, no flocculation was observed with MPEO–PLA particles at NaCl concentrations up to 5 M. Thus the formation of the MPEO layer leads to sterical stabilization by osmotic repulsion. 4. Conclusion Water-soluble MPEO–PLA copolymers with different MPEO and PLA block lengths were synthesized and were shown to act as nonionic water-soluble polymeric surfactants. The equilibrium surface tension depends mainly on the PLA block length and decreases with it. The area occupied by an adsorbed MPEO macromolecule indicates the formation of closely packed interfacial layers. Some of these MPEO–PLA copolymers may be used as stabilizers to produce stable hydrophilic PLA nanoparticles by the o/w emulsion/evaporation technique with a mean diameter of 200 nm. The area occupied per MPEO chains at the PLA particle surface is significantly higher than that of the same molecule at the water/air interface, meaning that the MPEO–PLA are less closely packed at the PLA particle surface. In a following paper, we will compare the in vivo cell interactions and biodistribution of the various MPEO–PLA particles in order to examine the effect of the MPEO layer architecture on the extent of phagocytosis. In addition, these copolymers will be used in a w/o/w emulsion process to encapsulate protein C. References [1] S. Stolnik, C.R. Heald, J. Neal, M.C. Garnett, S.S. Davis, L. Illum, S.C. Purkiss, R.J. Barlow, P.R. Gellert, J. Drug Target. 9 (2001) 361. [2] R. Gref, Y. Minamitake, M.T. Peracchia, V. Trubetskoy, V. Torchilin, R. Langer, Science 263 (1994) 1600.

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