Amphiphilic Polysaccharides Acting both as Stabilizers and Surface

Article www.acsabm.org

Cite This: ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

Amphiphilic Polysaccharides Acting both as Stabilizers and Surface Modifiers during Emulsification in Microfluidic Flow-Focusing Junction Asma Chebil,† Denis Funfschilling,‡,§ Michèle Léonard,† Jean-Luc Six,† Cécile Nouvel,†,∥ and Alain Durand*,† †

Université de Lorraine, CNRS, LCPM, F-54000 Nancy, France Université de Lorraine, CNRS, LRGP, F-54000 Nancy, France

Downloaded via UNIV OF READING on August 16, 2018 at 20:15:33 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

ABSTRACT: A continuous emulsion/solvent diffusion process was designed for the preparation of polysaccharide-covered poly(D,L-lactide) (PLA) microparticles. The emulsification step was carried out in a flow-focusing junction where ethyl acetate containing dissolved PLA was dispersed into an aqueous solution of hydrophobically modified dextran. It was demonstrated that poly(dimethylsiloxane) devices could be used for oil-in-water emulsion preparation provided that the microfluidic devices were preconditioned by simply circulating the aqueous phase containing the amphiphilic polysaccharide during a sufficient time (30 h). The adsorption of the polymers at the surface of the channel walls permitted the wetting by the aqueous phase with a hydrophilic character maintained at least throughout 2 months. The preconditioning time was significantly reduced by pretreating the microfluidic device with piranha solution and KOH solution during 15 min each before the circulation of the aqueous solution of dextran derivative. Dextran-covered PLA microparticle aqueous suspensions were produced with well-controlled size distribution. The suspensions could be lyophilized and reconstituted by retrieving the initial size distribution without adding any cryoprotectant. The reported procedure was used for preparing octyl gallate-loaded PLA microparticles. KEYWORDS: amphiphilic polysaccharide, microfluidics, flow-focusing, emulsion, microparticles, surface hydrophilization, encapsulation

1. INTRODUCTION Emulsion-solvent evaporation/diffusion (ESE/D) is a very common process for preparing polymeric nano- and microparticles for drug encapsulation and delivery applications.1−5 Briefly, the polymer forming the core of the particles is dissolved (with the drug to be encapsulated) into a waterimmiscible volatile organic solvent (dichloromethane and ethyl acetate, EA, are common examples). This organic solution is emulsified into an aqueous phase which contains a stabilizer. After emulsion preparation, the organic solvent is fully eliminated either by evaporation or by diffusion into a large excess of aqueous phase (in the case of solvents having sufficient miscibility with water like EA). Solvent diffusion or evaporation leads to the formation of polymeric particles whose size distribution is mainly influenced by the choice of emulsification operation (sonication, mechanical agitation···) as well as formulation variables (nature and amount of stabilizer) provided that other steps (like solvent evaporation or diffusion) have no additional effect.6 Microfluidic devices are efficient systems for the continuous production of welldefined nano- or micrometric dispersions.7−9 If microfluidic systems are used for the emulsification step, it is possible to produce suspensions of polymeric microparticles with narrow © XXXX American Chemical Society

and well-controlled particle size distributions. Narrow particle size distribution leads to an improved control of the kinetics of release as compared to suspensions of particles with broader size distribution.10 Nevertheless, surface properties of microfluidic devices are one determinant parameter, and their variation may induce instability in the characteristics of produced particles.10 The surface of particles is generally covered by a surfactant which prevents their aggregation during preparation, but other stabilizers like polymers could be used to confer other specific properties in view of applications (improved colloidal stability in brine, increased residence time by limitation of the action of the immune system, specific attachment of particles to some surfaces···). Previously, ESD involving emulsification in a Y-junction microfluidic device was designed to produce PLA microparticles covered by poly(ethylene glycol)-b-polylactide copolymer.11 We have particularly investigated the preparation of poly(lactic acid) (PLA) nanoparticles with well-defined surface characteristics using polymeric stabilizers derived from polysaccharides like dextran Received: July 8, 2018 Accepted: August 7, 2018 Published: August 8, 2018 A

DOI: 10.1021/acsabm.8b00303 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

Article

ACS Applied Bio Materials

glucose repeat units, expressed in percent. In that work, essentially one dextran derivative was used with τ = 17%. Poly(D,L-lactide) was purchase from Sigma-Aldrich. Its number-average molar mass M n = 48 000 g·mol−1 and dispersity Đ = 1.3 were determined by size exclusion chromatography using tetrahydrofuran as the eluent. 2.2. Fabrication of Microfluidic Flow Focusing Devices. PDMS flow focusing devices were fabricated using a soft standard photolithographic process.19 A silicon wafer was washed following a two-step procedure. In the first step, the wafer was dipped into acetone and then into a water bath with ultrasounds and finally dried. The second step involved dipping the wafer into ethanol and drying it. A layer of SU8 negative resin was deposited on the clean silicon wafer by spin coating and then backed at 95 °C during 45 min. This wafer was exposed to UV light through a mask and then developed with a specific solvent by dipping the wafer during 15−17 min under stirring. Then the wafer was washed with ethanol to remove the excess of resin and dried. The rest of the fabrication was carried out under a laminar flow hood. The previous SU8 master was covered with PDMS + 10% of curing agent (SYLGARD 184 Silicone Elastomer kit, Dow Corning) and then cured in an oven at 120 °C for 1 h. Finally, the PDMS was sealed to a borosilicate glass wafer after an oxygen plasma pretreatment (Harrick Plasma Cleaner, Ithaca, New York, USA). The flow focusing system was formed by rectangular channels (depth 47 μm, width 180 μm for aqueous phase inlet, 101 μm for the organic phase inlet, and 187 μm for the emulsion outlet, Figure 1).

(a nonionic bacterial polysaccharide) or hyaluronic acid (an anionic polysaccharide) during the emulsification step.12−14 Such polysaccharide-based stabilizers exhibited biocompatibility and conferred specific recognition by biological receptors (hyaluronic acid) or possessed reactive groups allowing further surface functionalization (dextran).14,15 To the best of our knowledge, there has been no report about any ESE/D process for the preparation of polysaccharide-covered PLA microparticles with diameters in the 1−100 μm range. We have showed that polysaccharide-covered PLA nanoparticles could find applications in targeted and controlled drug delivery.14−16 In another work, we showed that polysaccharide-covered PLA microparticles with diameters higher than 100 μm could be used as microcarriers for mesenchymal stem cell expansion.17 Thus, the use of microfluidic systems would nicely increase the range of particle diameters available with potential bioapplications. Such a strategy would allow improving the control of both size distribution and surface properties of final particles as compared to devices involving either ultrasounds or mechanical agitation for producing the intermediate emulsion. Thus, our aim was to design such a process, to investigate how the specific properties of amphiphilic polysaccharides could affect the operating conditions, and to characterize the obtained microparticles. The amphiphilic polysaccharides used in that work have been obtained by covalent attachment of hydrocarbon groups along dextran chains (6 carbon atoms, random distribution).18 Hydrocarbon groups randomly distributed along the polysaccharide backbone accumulated at the oil/ water interface, thus driving physical adsorption of amphiphilic polysaccharides at the surface of droplets/particles. Because of their structure, macromolecules formed loops of unmodified glucose units protruding into the aqueous phase separated by adsorbed alkylated units which produced a hydrophilic layer. As an example, thicknesses of superficial layers up to 10 nm have been measured for nanoparticles prepared in the presence of similar hydrophobically modified dextrans.12,16 In that work, we investigated the continuous preparation of dextran-covered PLA microparticles with diameters in the 10− 100 μm range, using ESD procedure and involving the flowfocusing microfluidic junction for the emulsification step. The presence of dextran chains at the surface of PLA microparticles was aimed at providing biocompatible surface coverage and, because of the high density of superficial reactive hydroxyl groups, was expected to be used for further surface functionalization. Furthermore, regarding the preparation process itself, the use of dextran-based polymeric stabilizers represented an additional opportunity for developing a new and easy to handle preconditioning procedure of the microfluidic system to control surface properties of channel walls. PLA microparticle suspensions were characterized by their particle size distribution and their ability for redispersion after washing and lyophilization. Finally, the reported procedure was applied to the preparation of octyl gallateloaded microparticles.

Figure 1. Experimental setup for microparticle production with a zoom on MFFD (left).

2.3. Setup for Fabrication of Microparticles. The flow rates of the aqueous and organic phases were regulated by syringe pumps (Havard Apparatus) with a double piston which allowed doubling the production capacity (Figure 1).10 The syringes (Hamilton) were in glass with pistons in PTFE. The formation of emulsions at the outlet of the flow focusing device was observed with a high-resolution camera (Canon EOS 70D equipped with the macroscopic canon zoom MP-ε 65 mm f/2.8 1−5×). At the inlets of the MFFD, tubes in PTFE were used for aqueous and organic phases. At the outlet of the microfluidic flow focusing device a glass tube was set to transfer the oil-in-water emulsion into a round-bottom flask containing water for dilution. When a tube in PTFE (instead of a glass tube) was used at the outlet of the MFFD, the emulsion coalesced in the tube, and no particles could be produced. 2.4. Fabrication of Microparticles. The continuous phase was composed of an aqueous solution of DexC6-τ (5 g·L−1) saturated with EA, and the disperse phase was composed of EA containing PLA (10 g·L−1). For the preparation of octyl gallate (OG)-loaded microparticles, the disperse phase was composed of EA containing PLA (8 g·L−1) and OG (2 g·L−1).

2. EXPERIMENTAL SECTION 2.1. Materials. Dextran T40 was purchased from Pharmacia (manufacturer data: M n 35−45 000 g·mol−1). The other chemicals were purchased from Sigma-Aldrich and used without further purification. Hydrophobically modifed dextrans were prepared and characterized as described previously.16 Dextran derivatives were named DexC6-τ in which τ was the substitution ratio defined as the mole ratio of attached hydrophobic groups to the total number of B


Article


Figure 2. General scheme of the process of fabrication of dextran-covered PLA microparticles. The surface of the MFFD walls (in PDMS) surface was preconditioned by circulating the aqueous phase during several hours prior to starting the fabrication of microparticles. Alternatively, piranha solution (mixture of 30 wt % H2O2 and H2SO4 1:2 in volume) was introduced into the MFFD using a syringe and let in contact for 15 min. The MFFD was washed with water during 5 min. Then following the same procedure, 1 M KOH solution was introduced into the MFFD and let in contact for 15 min followed by final rinsing with water during 5 min.20 Microparticle production could be started immediately after this treatment. At the outlet, EA was finally removed by solvent diffusion in a large volume of water.21 The resulting particles were filtered, washed with pure water to remove nonadsorbed DexC6-τ (1.2 μm mesh, MilliPore system), and redispersed into a minimal volume of water (10 mL). 2.5. Characterization of Microparticles. Particle size distribution was determined using laser light-scattering (Mastersizer 2000 Malvern Instruments-Cellule Hydro 2000 μp) experiments. The width of the size distribution was evaluated by the Span value (eq 1). Span = [(d(0.9) − d(0.1)]/d(0.5)

3. RESULTS AND DISCUSSION 3.1. Continuous Process for the Preparation of Dextran-Covered PLA Microparticles. A continuous ESD process for the production of dextran-covered microparticles was designed comprising three successive unit operations (Figure 2). The first unit operation was the production of oil-in-water micrometric emulsion into a flow-focusing microfluidic device (MFFD). As compared to T-junctions, MFFDs have been reported to lead to higher frequency of droplet formation and thus have been widely used for in situ droplet generation.22−24 A 10 g·L−1 solution of PLA in ethyl acetate (EA) flowed into the central channel, and an aqueous solution containing a hydrophobically modified dextran (DexC6-τ, see Experimental Section) was injected into the two side channels. Because of the significant solubility of EA in water (8.3% v/v),25 the aqueous phase was saturated with EA before introduction into the microfluidic device. The concentration of DexC6-τ in the aqueous phase was set to 5 or 10 g·L−1, so as to have a fast enough adsorption of amphiphilic polymers onto the surface of newly created droplets.18 For this first unit operation, there were three important working parameters that we investigated: the flow rates of aqueous (Qc) and organic (Qd) phases and the wetting properties of the channel walls.22 Once formed in the MFFD, oil-in-water emulsion was continuously transferred through a glass tube to the second unit operation where it was diluted in an excess of water to extract EA and thus lead to the solidification of the particle core. We tried to minimize the volume of water used for that operation (Vdilution) to limit the dilution of the final suspension. Knowing the solubility of EA in water, it was possible to calculate the minimum volume of water (Vmin) required for complete dissolution of the amount of EA corresponding to a given production of PLA particles. The volume ratio Vdilution/ Vmin was the main working parameter to be investigated for this second unit operation of the process. In the third unit operation, particle suspension was filtered on paper (1.2 μm porosity), and the recovered particles were extensively washed with Milli-Q water to extract any stabilizer in excess. During these operations, some losses of matter were

(1)

In eq 1, d(0.9), d(0.5), and d(0.1) were the diameters at 90%, 50%, and 10% cumulative volumes, respectively. The average particle diameter was evaluated either using d(0.5) or the surface-average diameter noted D[3,2]. Morphological examination of microparticles was performed by transmission electron microscopy (TEM) (Philips CM200, 200 kV). The amount of encapsulated OG per g of PLA was determined by 1 H NMR analysis (Bruker Avance 300 MHz) after complete dissolution of microparticles (previously extensively washed and lyophilized) in DMSO-d6. 2.6. Contact Angle Measurements. The effect of each surface treatment of the PDMS surface was characterized by determining the contact angle with pure water. PDMS samples were prepared in Petri dishes and contacted with different liquids (aqueous solution of DexC6-τ, piranha solution···) during precise times. After the required time, small pieces of treated PDMS were cut, rinsed with milli-Q water, and dried with nitrogen. Using a Hamilton syringe (10 μL), 5 μL drops of milli-Q water (with 18.4 MΩ.cm resistivity) were deposited onto pieces of PDMS. Images of drops were taken using a high-resolution camera (Canon EOS 70D equipped with the macroscopic canon zoom MP-ε 65 mm f/2.8 1−5×). Static contact angle was estimated using the software called Protractor with the exploitation system IOS.20 At least 7 measurements were carried out for each condition of treatment, corresponding to different pieces of the same PDMS sample. The experimental accuracy was ±2°. C


Article


Figure 3. (Left) Contact angle measurement of milli-Q water drops onto the PDMS surface treated during at different times by contacting with 5 g· L−1 of DexC6-17 aqueous solution saturated with EA without (open circles) or after 15 min contacting with piranha solution and 15 min contacting 1 M KOH solution (bold circles). For details see the Experimental Section. Insets: images of water drops on untreated PDMS (a) or PDMS treated during 30 h (b and c). (Right) Schematic representation of the surface of PDMS after different treatments.

Figure 4. Images of MFFDs during microparticle production after different surface treatment. (A) Preconditioning by 1 h circulation of DexC6-17 (10 g·L−1) aqueous solution saturated with EA. (B) Preconditioning by 30 h circulation of DexC6-17 (10 g·L−1) aqueous solution saturated with EA. (C) Same treatment as B but image of the emulsion well after the MFFD. The organic phase was EA containing 10 g·L−1 of PLA, and the flow rates were Qc = 10 mL·h−1 and Qd = 0.1 mL·h−1.

difficult to avoid. They could be limited by minimizing the volume of water (Vdilution) used for extracting EA in the second unit operation. After the last filtration, particles were dispersed into 10 mL of water for the final characterization. For storage, the suspensions were lyophilized. When necessary, the amount of suspension produced was increased by using two MFFD and two dilution units in parallel.22 The characteristics of the suspensions were not affected by doubling the production line, which showed the possibility of scale up of the production. 3.2. Simple Hydrophilization of PDMS Microfluidic Systems by Preconditioning with Amphiphilic Polysaccharide Solution. PDMS was used for the fabrication of microfluidic devices. It is the most common material because of its many advantages: cheap, transparent, biocompatible, and easy to handle, etc.19,26−28 Nevertheless, for the production of aqueous dispersions its hydrophobicity was a strong drawback. Indeed, in order to produce oil-in-water emulsions, it was required that the channels were wetted by the continuous aqueous phase.29 Thus, hydrophilization of PDMS channel walls was required. This point has been widely investigated, and several strategies have been reported: plasma treatment, layer-by-layer deposition, chemical vapor deposition, surfactant addition into the aqueous phase, incorporation of commercial amphiphilic block copolymers (Pluronic F127) into the PDMS

prepolymer before curing, preliminary physical adsorption of protein, etc.29−35 Most of these treatments, even those that are easy to apply, have to face with the difficulty that the PDMS surface progressively recovered its hydrophobicity, which limited the time of usability of the microfluidic device. The main reasons proposed for explaining this reversibility of hydrophilization were: the mobility of PDMS chains which rearranged and reoriented hydrophilic groups toward the bulk PDMS and the migration of PDMS oligomers from the bulk toward the surface of the material.20,30,36 Taking into account the amphiphilic properties of DexC6-τ dextran derivatives, a first series of experiments were carried out in order to evaluate the possible adsorption of the stabilizer onto the channel walls and the resulting modification of surface energy. Water contact angle measurements were carried out on PDMS plates immersed into a 5 g·L−1 DexC6-17 aqueous solution (saturated with EA) during various time intervals (Figure 3). After 30 h of impregnation, the contact angle dropped down to 101.8° instead of 111.8° for the native PDMS. The decrease of contact angle further continued, leading roughly to a minimum value of 80−85°. The physical adsorption of DexC6-τ was expected to cover the PDMS surface by a hydrophilic layer formed by dextran loops (Figure 3). D


Article


experimental demonstration of its suitability for the preparation of O/W emulsion in PDMS microsystems. Contact angle measurements showed that the combination of chemical treatment and DexC6-17 adsorption provided a rapid hydrophilic character to the PDMS surface which was maintained over 2 months (Figure 3). The same treatment was then applied to MFFD. MFFD was treated by injection of piranha solution followed by 15 min contact without circulation. After rinsing with Milli-Q water during 5 min, 1 M KOH solution was injected and let in static contact during 15 min. After final washing by circulating Milli-Q water during 5 min, the MFFD was used for ESD. For all these experiments the DexC6-17 concentration was kept equal to 5 g·L−1. An O/ W emulsion was obtained with isometric droplets showing that this low cost, simple, and fast (40 min) treatment led to readyto-use MFFD. In addition, if the MFFD was kept in contact with the 5 g·L−1 DexC6-17 aqueous solution, it remained usable for particle preparation over more than one month. In what follows, this treatment was applied to MFFD for particle preparation. 3.3. Production and Characteristics of DextranCovered PLA Microparticles. For the emulsification step, the flow rates, Qc and Qd, were the investigated working parameters. The formation of isometric droplets was possible in the dripping regime.43 The experimental ranges of values were 0.1 to 0.3 mL·h−1 for Qd and 4 to 10 mL·h−1 for Qc. With these previous values, the dripping regime was experimentally observed. In order to justify this observation, the capillary numbers, Cac and Cad, were defined according to eq 2.

Starting from these experiments, several MFFDs were preconditioned by circulating an aqueous solution of DexC617 during several time intervals (Figure 4). With the aim of reducing the time needed for this preconditioning step, the DexC6-17 concentration was increased up to 10 g·L−1 (as compared to 5 g·L−1 for contact angle measurements). For the shortest preconditioning time (1 h), emulsification was not possible in the MFFD, and it was observed that the EA phase was wetting the channels. This was attributed to an insufficient time for significant DexC6-17 adsorption and thus modification of the surface hydrophilicity. On the contrary, after 30 h preconditioning, emulsification occurred, and oil droplets were formed for Qc = 10 mL·h−1 and Qd = 0.1 mL·h−1 (Figure 4). This result was fully consistent with our contact angle decreasing measurements on the basis of a previous systematic study of the effect of surface energy of PDMS walls on emulsification in flow-focusing devices.29 The authors reported that when the contact angle of water on the channel walls was equal to 105° and 112° no oil-in-water emulsion formed. On the contrary, when the contact angle of water on the channel walls was equal to 92° oil droplets were produced. In addition, our experiments showed that a minimum time (of the order of several hours) was required for preconditioning to reach a sufficient adsorption of amphiphilic dextran derivatives. As a conclusion, provided that the preconditioning step was long enough, MFFD could be used for O/W emulsion preparation without any PDMS treatment except contacting the MFFD with the aqueous solution of the amphiphilic dextran derivative. To the best of our knowledge, it was the first time that a polymeric stabilizer involved in the ESD process was used for hydrophilization of PDMS channels. This procedure has several advantages since it did not require modifying the procedure of fabrication of MFFD; it did not involve any other reactant than those used for the preparation of microparticles; and it could be carried out by a simple circulation of the aqueous solution before starting the ESD process. Despite its simplicity, one main drawback of preconditioning MFFD with 10 g·L−1 of DexC6-17 aqueous solution was the time needed before starting the ESD process. Further increase of DexC6-17 concentration may allow decreasing the time needed but would require excessively high amounts of amphiphilic polymer. In order to shorten that time, we proposed to combine the rather slow physical adsorption of DexC6-17 with a comparatively fast chemical treatment of MFFD. Our assumption was that the chemical treatment would provide rapid hydrophilization of the PDMS surface, while the slower adsorption of DexC6-17 would bring a longterm hydrophilic character to channel walls. Indeed, even if the effect of the chemical was progressively lost because of rearrangement of superficial groups, the quasi-irreversible adsorption of DexC6-17 would maintain the hydrophilic characteristic of the MFFD walls over a long time. We used a simple and fast chemical treatment consisting of 15 min exposure of PDMS to piranha solution followed by 15 min exposure to KOH solution.20 This treatment has been shown to improve efficiently and rapidly PDMS surface hydrophilicity which was explained by an increase of hydroxyl groups present at the surface of the material (Figure 3). Nevertheless, the hydrophobic character was recovered within 24 h when the sample was stored under vacuum. Although this treatment has been suggested for microfluidic devices,33,37−42 to the best of our knowledge our work was the first

Cac =

μc Uc γcd

Cad =

μd Ud γcd

(2)

In eq 2, μc and μd were the viscosities (Pa·s); Uc and Ud were the superficial velocities (m·s−1) of the continuous and dispersed phase, respectively; and γcd was the interfacial tension (N·m−1). In the aqueous phase, because of the concentration of DexC6-17, the viscosity was assumed to be identical to that of pure water on the basis of previous results.18 In ethyl acetate, the intrinsic viscosity of PLA was estimated to be 0.071 L·g−1 from literature data.44 Thus, the viscosity of the organic phase was estimated as twice that of EA, i.e., 0.85 mPa· s.45 Without experimental data about the interfacial tension, γcd, it was replaced by the value between pure liquids, i.e., 0.0068 N·m−1. With those values, within the investigated ranges of Qd and Qc, Cac was between 0.010 and 0.024, and Cad was between 0.0007 and 0.002. These values corresponded to the dripping regime for this flow-focusing geometry.43,46 As for the dilution step, the volume ratio Vdilution/Vmin was varied, everything else being equal. We found that narrow particle size distributions were obtained for a ratio equal to 3. This result was consistent with those of Imbrogno et al., who reported a two-step ESD process for the preparation of polycaprolactone microparticles involving membrane emulsification followed by solvent dilution in water.47 This value of 3 was kept for all experiments. Several samples of particle suspensions were prepared using previously established conditions (Table 1). The relevant parameter controlling the characteristics of particles size distribution was the ratio of flow rates (Qc/Qd). For Qc/Qd ratios higher than 20, narrow particle size distributions were obtained with Span values lower than 1. Doubling both flow E


Article


lower than the diameter of the droplets produced during emulsification. Droplets formed at the outlet of MFFD had diameters close to 100 μm, and the recovered PLA microparticles had diameters close to 20 μm, i.e., in perfect agreement with eq 3. After the dilution step, particles were recovered by filtration on paper, extensively washed with pure water, and finally dispersed in pure water. The particle size distribution was not modified by this last operation. TEM images of dextrancovered PLA microparticles confirmed the spherical shape of the particles as well as the size determined by laser granulometry (Figure 6). Since storage of aqueous suspensions presented serious drawbacks (possible degradation, volume, etc.), the possibility to lyophilize the suspensions and redisperse them by simple water addition was investigated. Lyophilized particles could be fully redispersed in water, and the particle size distribution was identical to that of initial suspension (Figure 7). No cryoprotectant was used. This excellent redispersion ability was due to the presence of amphiphilic polysaccharide at the surface of particles. Adsorbed macromolecules formed a thick (between 5 and 10 nm had been measured in the case of PLA nanoparticles) hydrophilic layer which prevented particle irreversible aggregation. TEM images provided a supplementary proof of this easy and complete redispersion of lyophilized suspension (Figure 7).12 3.4. Encapsulation of Octyl Gallate into DextranCovered PLA Microparticles. As in our previous report about nanoparticles, OG was selected as a model hydrophobic drug. OG has been shown to exhibit miscibility with PLA in the solid state up to 30 wt % of OG in the OG + PLA mixture.16 Consequently, we chose to carry out encapsulation experiments with OG + PLA mixtures containing 20 wt % of OG. The process conditions were the same as in section 3.3 with Qc = 6 mL·h−1 and Qd = 0.3 mL·h−1 except that the organic phase was replaced by EA containing PLA (8 g·L−1) and OG (2 g·L−1). Our results demonstrated that the continuous process could be applied for encapsulating hydrophobic molecules into dextran-covered PLA microparticles. The size of the microparticles was not significantly modified by the presence of OG in the organic phase (Table 2). Moreover, OG-loaded microparticle suspensions could be extensively washed, lyophilized, and redispersed while keeping their size distribution, as in the case of bulk PLA microparticles.

Table 1. Characteristics of Oil Droplets and PLA Microparticles Obtained with Different Flow Rates of the Aqueous Phase (5 g·L−1 DexC6-17) and Organic Phase (10 g·L−1 PLA in EA) Qc/Qd (mL·h−1)/(mL·h−1)

4.0/0.3

5.0/0.3

6.0/0.3

8.0/0.2

d(0.5) (μm)a spana Dg (μm)b calculated particle diameter (μm)c

6 7.5 103 20

28 1.3 96 19

22 0.7 86 17

21 0.5 80 15

a

Measured by laser granulometry. bAverage droplet diameter determined by image analysis using MatLab with approximately 20 droplets. cCalculated values using average droplet diameter and eq 3.

rates (keeping unchanged Qc/Qd ratio) led to almost identical particle size distributions (Figure 5).

Figure 5. Particle size distributions (in volume by laser granulometry) of microparticles recovered after filtration and washings by changing the flow rates of both phases (Qc and Qd as indicated on the graph) while keeping a Qc/Qd ratio equal to 40.

Assuming that each oil droplet would lead to one single solid particle after solvent diffusion, a direct link between droplet diameter (Dg) and particle diameter (Dp) was expected (eq 3).

ij yz jj zz jj zz 1 j zz Dp = Dg jj jj zz 1 − x PLA ρPLA z z jj 1 + xPLA ρAE z (3) k { In eq 3, xPLA was the mass fraction of PLA in the oil droplet, and ρPLA and ρAE were the densities of PLA and EA (g·mL−1), respectively. With the concentration of PLA in EA equal to 10 g·L−1, it was thus estimated that particle diameter should be 5 times 1/3

( )

Figure 6. Image of MFFD system (left), particle size distribution (in volume by laser granulometry) of suspensions after dilution and after filtration and washings (as indicated on the graph), and TEM micrograph of particles after filtration and washings. For this experiment Qc = 6 mL·h−1 and Qd = 0.3 mL·h−1 (see Table 1 for other conditions). F


Article


Figure 7. (Left) Particle size distributions (in volume by laser granulometry) of microparticles recovered after dilution and after lyophilization and reconstitution (as indicated on the graph). For this experiment Qc = 6 mL·h−1 and Qd = 0.3 mL·h−1 (see Table 1 for other conditions). (Right) TEM micrograph of particles after lyophilization.

lyophilization of the suspensions without using any cryoprotectant. The suspensions could be stored in a dry state and easily redispersed before use. Thus the use of dextran-based polymeric surfactants appeared particularly attractive for microparticle preparation involving MFFD both regarding process conditions and product properties. The current work deals with improvement of encapsulation performances of model hydrophobic drugs into PLA microparticles produced by this process and the study of the corresponding kinetics of release.

Table 2. Characteristics of PLA and PLA + OG Microparticles Obtained with 6.0 mL·h−1 of Aqueous Phase (5 g·L−1 DexC6-17) and 0.3 mL·h−1 of Organic Phase (Either 10 g·L−1 of PLA in EA or 8 g·L−1 of PLA + 2 g·L−1 of OG in EA) OGa

D[3,2]b (μm)

(wt %)

before washing

after washing

after lyophilization and redispersion

loaded OGc (wt %)

0 20

22 27

24 30

23 28

0 3.0

■

a

In the organic phase at the inlet of the microfluidic device. bSurfaceaverage diameter of microparticles measured by laser granulometry. c Determined by 1H NMR analysis of the recovered microparticles (see Experimental Section for details).

AUTHOR INFORMATION

Corresponding Author

*Tel.: + 33 (0)3 72 74 37 01. Fax: + 33 (0)3 83 37 99 77. Email: [email protected]. ORCID

The relatively low encapsulated amount of OG (3 wt % in the final microparticles as compared to 20 wt % at the inlet) could be attributed to the increased solubility of OG in the aqueous phase because of the presence of DexC6-τ, which has been already raised in the case of encapsulation into nanoparticles.16 Further experimental work would be necessary to improve that point.

Jean-Luc Six: 0000-0003-1151-6953 Alain Durand: 0000-0003-1017-6029 Present Addresses §

Université de Strasbourg, CNRS, ICube, F-67000 Strasbourg, France. ∥ Université de Lorraine, CNRS, LRGP, F-54000 Nancy, France.

4. CONCLUSIONS Dextran-covered PLA microparticles with narrow size distributions have been produced using an emulsion-solvent diffusion process which involved emulsification in a PDMS flow-focusing device. Amphiphilic dextran derivatives were shown to act as both stabilizers during emulsification step and surface modifiers for PDMS channel walls conferring hydrophilicity to the surface of the material. Preconditioning the flow-focusing device by circulating a 5 g·L−1 aqueous solution of amphiphilic dextran during 30 h allowed using MFFD for O/W emulsion preparation without any other treatment. The preconditioning step could be replaced by a faster and simple chemical treatment using piranha and KOH solutions. This chemical treatment allowed starting the production of microparticles immediately, while the presence of amphiphilic dextran derivatives in the external aqueous phase extended the hydrophilicity of PDMS over more than one month. This preconditioning procedure was fast (only 40 min) and very easy to handle, required only contacting the device with solutions, and provided surface hydrophilicity which was maintained over a long time. To the best of our knowledge, this preconditioning procedure has never been reported. It could be generalized to many other systems designed for formulating microparticles. Dextran-covered PLA microparticles exhibited good stability to extensive washings and excellent redispersion ability after

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support of Région Lorraine and European Union (FEDER) to the BioCapTech project (PhD Thesis of Asma Chebil). Loic̈ Chomel de Varagnes and Jean-Claude Pihan (BioCapTech Company) are acknowledged for many fruitful discussions.

■

REFERENCES

(1) Mora-Huertas, C. E.; Fessi, H.; Elaissari, A. Influence of Process and Formulation Parameters on the Formation of Submicron Particles by Solvent Displacement and Emulsification−Diffusion Methods Critical Comparison. Adv. Colloid Interface Sci. 2011, 163, 90−122. (2) Bodmeier, R.; McGinity, J. W. The Preparation and Evaluation of Drug-Containing Poly(dl-lactide) Microspheres Formed by the Solvent Evaporation Method. Pharm. Res. 1987, 4, 465−471. (3) O’Donnell, P. B.; McGinity, J. W. Preparation of Microspheres by the Solvent Evaporation Technique. Adv. Drug Delivery Rev. 1997, 28, 25−42. (4) Lassalle, V.; Ferreira, M. L. PLA Nano- and Microparticles for Drug Delivery: An Overview of the Methods of Preparation. Macromol. Biosci. 2007, 7, 767−783. (5) Anton, N.; Benoit, J.-P.; Saulnier, P. Design and Production of Nanoparticles Formulated from Nano-Emulsion TemplatesA Review. J. Controlled Release 2008, 128, 185−199.

G


Article

ACS Applied Bio Materials (6) Desgouilles, S.; Vaulthier, C.; Bazile, D.; Vacus, J.; Grossiord, J.L.; Veillard, M.; Couvreur, P. The Design of Nanoparticles Obtained by Solvent Evaporation: A Comprehensive Study. Langmuir 2003, 19, 9504−9510. (7) Whitesides, G. M. The Origins and the Future of Microfluidics. Nature 2006, 442, 368−373. (8) Zhao, C.-X. Multiphase Flow Microfluidics for the Production of Single or Multiple Emulsions for Drug Delivery. Adv. Drug Delivery Rev. 2013, 65, 1420−1446. (9) Shim, T. S.; Kim, S.-H.; Yang, S.-M. Elaborate Design Strategies Toward Novel Microcarriers for Controlled Encapsulation and Release. Particle 2013, 30, 9−45. (10) Xu, Q.; Hashimoto, M.; Dang, T. T.; Hoare, T.; Kohane, D. S.; Whitesides, G. M.; Langer, R.; Anderson, D. G. Preparation of Monodisperse Biodegradable Polymer Microparticles Using a Microfluidic Flow-Focusing Device for Controlled Drug Delivery. Small 2009, 5, 1575−1581. (11) Watanabe, T.; Ono, T.; Kimura, Y. Continuous Fabrication of Monodisperse Polylactide Microspheres by Droplet-to-Particle Technology Using Microfluidic Emulsification and Emulsion−Solvent Diffusion. Soft Matter 2011, 7, 9894−9897. (12) Rouzes, C.; Léonard, M.; Durand, A.; Dellacherie, E. Influence of Polymeric Surfactants on the Properties of Drug-Loaded PLA Nanospheres. Colloids Surf., B 2003, 32, 125−135. (13) Léonard, M.; Marie, E.; Wu, M.; Dellacherie, E.; Camesano, T.; Durand, A. Preparation of Polysaccharide-Covered Polymeric Nanoparticles by Several Processes Involving Amphiphilic Polysaccharides. ACS Symp. Ser. 2008, 996, 322−340. (14) Laroui, H.; Grossin, L.; Leonard, M.; Stoltz, J.-F.; Gillet, P.; Netter, P.; Dellacherie, E. Hyaluronate-Covered Nanoparticles for the Therapeutic Targeting of Cartilage. Biomacromolecules 2007, 8, 3879− 3885. (15) Zille, H.; Paquet, J.; Henrionnet, C.; Scala-Bertola, J.; Léonard, M.; Six, J.-L.; Deschamps, F.; Netter, P.; Verges, J.; Gillet, P.; Grossin, L. Evaluation of Intra-Articular Delivery of Hyaluronic Acid Functionalized Biopolymeric Nanoparticles in Healthy Rat Knees. Bio-Med. Mater. Eng. 2010, 20, 235−242. (16) Chebil, A.; Léonard, M.; Six, J.-L.; Nouvel, C.; Durand, A. Nanoparticulate Delivery Systems for Alkyl Gallates: Influence of the Elaboration Process on Particle Characteristics, Drug Encapsulation and In-Vitro Release. Colloids Surf., B 2018, 162, 351−361. (17) Roy, A.; Murcia Valderrama, M. A.; Daujat, V.; Ferji, K.; Léonard, M.; Durand, A.; Babin, J.; Six, J.-L. Stability of a Biodegradable Microcarrier Surface: Physically Adsorbed versus Chemically Linked Shells. J. Mater. Chem. B 2018, 6, 5130. (18) Rotureau, E.; Leonard, M.; Marie, E.; Dellacherie, E.; Camesano, T. A.; Durand, A. From Polymeric Surfactants to Colloidal Systems (1): Amphiphilic Dextrans for Emulsion Preparation. Colloids Surf., A 2006, 288, 131−137. (19) McDonald, J. C.; Duffy, D. C.; Anderson, J. R.; Chiu, D. T.; Wu, H.; Schueller, O. J. A.; Whitesides, G. M. Fabrication of Microfluidic Systems in Poly(dimethylsiloxane). Electrophoresis 2000, 21, 27−40. (20) Maji, D.; Lahiri, S. K.; Das, S. Study of Hydrophilicity and Stability of Chemically Modified PDMS Surface Using Piranha and KOH Solution. Surf. Interface Anal. 2012, 44, 62−69. (21) Fu, T.; Ma, Y.; Funfschilling, D.; Zhu, C.; Li, H. Z. Breakup Dynamics of Slender Bubbles in Non-Newtonian Fluids in Microfluidic Flow-Focusing Devices. AIChE J. 2012, 58, 3560−3567. (22) Seemann, R.; Brinkmann, M.; Pfohl, T.; Herminghaus, S. Droplet Based Microfluidics. Rep. Prog. Phys. 2012, 75, 016601. (23) Yobas, L.; Martens, S.; Ong, W.-L.; Ranganathan, N. HighPerformance Flow-Focusing Geometry for Spontaneous Generation of Monodispersed Droplets. Lab Chip 2006, 6, 1073−1079. (24) Ong, W.-L.; Hua, J.; Zhang, B.; Teo, T.-Y.; Zhuo, J.; Nguyen, N.-T.; Ranganathan, N.; Yobas, L. Experimental and Computational Analysis of Droplet Formation in a High-Performance Flow-Focusing Geometry. Sens. Sens. Actuators, A 2007, 138, 203−212.

(25) Grande, M. d. C.; Marschoff, C. M. Liquid-Liquid Equilibria for Water + Benzonitrile + Ethyl Acetate or + Butyl Acetate. J. Chem. Eng. Data 2005, 50, 1324−1327. (26) Blake, A. J.; Pearce, T. M.; Rao, N. S.; Johnson, S. M.; Williams, J. C. Multilayer PDMS Microfluidic Chamber for Controlling Brain Slice Microenvironment. Lab Chip 2007, 7, 842−849. (27) Azmayesh-Fard, S. M.; Flaim, E.; McMullin, J. N. PDMS Biochips with Integrated Waveguides. J. Micromech. Microeng. 2010, 20, 087002. (28) Fleger, M.; Neyer, A. PDMS Microfluidic Chip with Integrated Waveguides for Optical Detection. Microelectron. Eng. 2006, 83, 1291−1293. (29) Li, W.; Nie, Z.; Zhang, H.; Paquet, C.; Seo, M.; Garstecki, P.; Kumacheva, E. Screening of the Effect of Surface Energy of Microchannels on Microfluidic Emulsification. Langmuir 2007, 23, 8010−8014. (30) Makamba, H.; Kim, J. H.; Lim, K.; Park, N.; Hahn, J. H. Surface Modification of Poly(dimethylsiloxane) Microchannels. Electrophoresis 2003, 24, 3607−3619. (31) Zhou, J.; Ellis, A. V.; Voelcker, N. H. Recent Developments in PDMS Surface Modification for Microfluidic Devices. Electrophoresis 2010, 31, 2−16. (32) Zhou, J.; Khodakov, D. A.; Ellis, A. V.; Voelcker, N. H. Surface Modification for PDMS-Based Microfluidic Devices. Electrophoresis 2012, 33, 89−104. (33) Gitlin, L.; Schulze, P.; Ohla, S.; Bongard, H.-J.; Belder, D. Surface Modification of PDMS Microfluidic Devices by Controlled Sulfuric Acid Treatment and the Application in Chip Electrophoresis. Electrophoresis 2015, 36, 449−456. (34) Sun, L.; Luo, Y.; Gao, Z.; Zhao, W.; Lin, B. Easy-to-Fabricate Thin-Film Coating on PDMS Substrate with Super Hydrophilicity and Stability. Electrophoresis 2015, 36, 889−892. (35) Wang, L.; Sun, B.; Ziemer, K. S.; Barabino, G. A.; Carrier, R. L. Chemical and Physical Modifications to Poly(dimethylsiloxane) Surfaces Affect Adhesion of Caco-2 cells. J. Biomed. Mater. Res., Part A 2010, 93A, 1260−1271. (36) Eddington, D. T.; Puccinelli, J. P.; Beebe, D. J. Thermal Aging and Reduced Hydrophobic Recovery of Polydimethylsiloxane. Sens. Sens. Actuators, B 2006, 114, 170−172. (37) Kalinova, R.; Mincheva, R.; Dubois, P. Imparting Adhesion Property to Silicone Materials: Challenges and Solutions. Rev. Adhesion Adhesives 2014, 2, 30−55. (38) Kowalewska, A.; Nowacka, M. In Concise Encyclopedia of High Performance Silicones; Tiwari, A., Soucek, M. D., Eds.; Scrivener Publishing LLC: 2014; pp 371−380. (39) Hemmilä, S.; Cauich-Rodriguez, J. V.; Kreutzer, J.; Kallio, P. Rapid, Simple, and Cost-Effective Treatments to Achieve Long-Term Hydrophilic PDMS Surfaces. Appl. Surf. Sci. 2012, 258, 9864−9875. (40) Li, G.; Parmar, M.; Lee, D.-W. An Oxidized Liquid Metal-Based Microfluidic Platform for Tunable Electronic Device Applications. Lab Chip 2015, 15, 766−775. (41) Yang, L.; Hao, X.; Wang, C.; Zhang, B.; Wang, W. Rapid and Low Cost Replication of Complex Microfluidic Structures with PDMS Double Casting Technology. Microsyst. Technol. 2014, 20, 1933−1940. (42) Zhuang, Q.; Li, T.; Lv, H.; Zhang, H.; Zhao, T. Stable and Homogenous Functionality on PDMS Surface and the Kinetic of Gold Nanoparticle Adsorption on Its Surface. Soft Mater. 2014, 12, 334−338. (43) Carrier, O.; Ergin, F. G.; Li, H.-Z.; Watz, B. B.; Funfschilling, D. Time-Resolved Mixing and Flow-Field Measurements During Droplet Formation in a Flow-Focusing Junction. J. Micromech. Microeng. 2015, 25, 084014. (44) Xu, K.; Kozluca, A.; Denkbas, E. B.; Piskin, E. Synthesis of PDLLA Homopolymers with Different Molecular Weights. J. Appl. Polym. Sci. 1996, 59, 561−563. (45) Pires, R. M.; Costa, H. F.; Ferreira, A. G. M.; Fonseca, I. M. A. Viscosity and Density of Water + Ethyl Acetate + Ethanol Mixtures at H


Article

ACS Applied Bio Materials 298.15 and 318.15 K and Atmospheric Pressure. J. Chem. Eng. Data 2007, 52, 1240−1245. (46) Carrier, O. PhD Thesis, Université de Lorraine, Nancy, France, 2012. (47) Imbrogno, A.; Piacentini, E.; Drioli, E.; Giorno, L. Preparation of Uniform Poly-Caprolactone Microparticles by Membrane Emulsification/Solvent Diffusion Process. J. Membr. Sci. 2014, 467, 262−268.

I


Amphiphilic Polysaccharides Acting both as Stabilizers and Surface

Recommend Documents