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Improvement of an encapsulation process for the preparation of pro- and prebiotics-loaded bioadhesive microparticles by using experimental design
European Journal of Pharmaceutical Sciences 44 (2011) 83–92
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European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps
Improvement of an encapsulation process for the preparation of pro- and prebiotics-loaded bioadhesive microparticles by using experimental design D. Pliszczak a,b,⇑, S. Bourgeois b,2, C. Bordes a,1, J.P. Valour b,2, M.A. Mazoyer c, A.M. Orecchioni d, E. Nakache e, P. Lantéri a a
Université de Lyon, F-69622, Lyon, France; Université Lyon 1, Villeurbanne; Laboratoires des sciences analytiques (LSA) UMR 5180, CNRS, CPE, 43 bd du 11 novembre 1918, 69100 Villeurbanne, France Université de Lyon, F-69622, Lyon, France; Université Lyon 1, Villeurbanne; Laboratoire d’Automatique et de Génie des Procédés (LAGEP), ISPB, UMR 5007, CNRS, CPE, 43 bd du 11 novembre 1918, 69100 Villeurbanne, France c Département pédagogique des sciences biomédicales A, ISPB faculté de pharmacie, Université Lyon 1, France d UFR de Médecine et Pharmacie, Université de Rouen, 22 boulevard Gambetta, 76000 Rouen, France e LCMT, UMR CNRS 6507, ENSICAEN, 6 Boulevard Maréchal Juin, 14000 CAEN, France b
a r t i c l e
i n f o
Article history: Received 4 April 2011 Received in revised form 15 June 2011 Accepted 19 June 2011 Available online 25 June 2011 Keywords: Vaginal delivery system Pectin Hyaluronic acid Probiotics Prebiotics Bioadhesive microparticles
a b s t r a c t The purpose of this study was to design a new vaginal bioadhesive delivery system based on pectinate– hyaluronic acid microparticles for probiotics and prebiotics encapsulation. Probiotic strains and prebiotic were selected for their abilities to restore vaginal ecosystem. Microparticles were produced by emulsification/gelation method using calcium as cross-linking agent. In the first step, preliminary experiments were conducted to study the influence of the main formulation and process parameters on the size distribution of unloaded microparticles. Rheological measurements were also performed to investigate the bioadhesive properties of the gels used to obtain the final microparticles. Afterwards an experimental design was performed to determine the operating conditions suitable to obtain bioadhesive microparticles containing probiotics and prebiotics. Experimental design allowed us to define two important parameters during the microencapsulation process: the stirring rate during the emulsification step and the pectin concentration. The final microparticles had a mean diameter of 137 lm and allowed a complete release of probiotic strains after 16 h in a simulated vaginal fluid at +37 °C. Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction The vagina is a large cavity which offers a potential route of administration for a local or a systemic drug delivery. Traditionally, solutions, suppositories, tablets, sponges, gels and foams have been used as vaginal formulations. The most common use is for a local delivery, especially for female-related conditions as antimicrobial, antifungal, antiviral, spermicidal agents, anti-inflammatory, prostaglandins and steroids (Hussain and Ahsan, 2005; Baloglu et al., 2009). According to the delivery system used, drug absorption, distribution and residence time in the vaginal tract may be different (Alambar and Akhrul, 2005). For example foams, solutions and suspensions allowed obtaining better coverage of vaginal tissue than ⇑ Corresponding author. Université de Lyon, F-69622, Lyon, France; Université Lyon 1, Villeurbanne; Laboratoires des sciences analytiques (LSA) UMR 5180, CNRS, CPE, 43 bd du 11 novembre 1918, 69100 Villeurbanne, France. Tel: +33 0 472448121/31844. E-mail address: [email protected] (D. Pliszczak). 1 Tel: +33 0 472448121. 2 Tel : +33 0 472431844 0928-0987/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2011.06.011
tablets (Hussain and Ahsan, 2005). Nonetheless liquid formulations are inappropriate for controlled drug release due to their short residence time in the vaginal tract. To obtain a local effect, semi-solid or fast dissolving solid bioadhesive system is required so as to uniformly distribute drug inside the vagina. Vaginal inserts and tablets allowed obtaining a continuous drug release for several hours (Alambar and Akhrul, 2005; Hussain and Ahsan, 2005; Baloglu et al., 2009). Particulate-delivery systems, as for example microspheres and liposomes, have been used for local or systemic action to deliver various drugs such as: acyclovir (Pavelic´ et al., 2005), calcitonin (Richardson et al., 1995, 1996; Rochira et al., 1996), insulin (Richardson et al., 1992), or acriflavine (Gavini et al., 2002). The main problem for vaginal drug delivery is the presence of cervical mucus which could rapidly remove delivery systems. Therefore physical factors of the vaginal tract have to be considered since many variations as cyclic changes, thickness of vaginal mucosa, properties of the vaginal fluid and sexual activity could affect drug delivery across the vaginal epithelium (Valenta, 2005). To improve the residence time of delivery systems in the vagina, mucoadhesive formulations could be developed using polymers as alginate, hyaluronic acid and derivatives, pectin,
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chitosan. . .which could interact with mucus by different mechanisms (Peppas and Buri, 1985; Gu et al., 1988; Woodley, 2001). Mucoadhesion phenomena are not well understood (Edsman and Hägerström, 2005; Smart, 2005) and several theories have been proposed to explain mucoadhesion (Peppas and Sahlin, 1996; Ahuja et al., 1997; Edsman and Hägerström, 2005; Khutoryanskiy, 2010) such as electronic, adsorption, wetting, fracture, mechanical and diffusion theories. Drug absorption and mucoadhesion efficiency are dependent on the physicochemical properties of the polymers and the drugs used in the formulation (Gurny et al., 1984; Duchene et al., 1988; Alambar and Akhrul, 2005; Asane et al., 2008). The use of probiotics, defined as ‘‘live microorganisms which when administered in adequate amounts confer a health benefit on the host’’ (Reid and Bruce, 2003; FAO/WHO, 2001) offers a potential alternative approach to health restoration and maintenance in the vaginal tract (Reid, 2001). Another way to operate consists in using prebiotics in order to provide nutrients which stimulate the Lactobacilli growth to the detriment of pathogens. The use of prebiotics is a more recently introduced concept. Many studies have shown prebiotics benefits on human health not only on gastrointestinal microflora but also anticancer and immune effects (Macfarlane et al., 2006; Lenoir-Wijnkoop et al., 2007). Rousseau et al. (2005) studied prebiotic effects on the urogenital ecosystem. They evaluated fructo- and gluco-oligosaccharides series varying by their osidic linkages. The results showed that oligosaccharides are good candidates to prevent vaginal infections. To our knowledge, nowadays no pharmaceutical studies were described for the encapsulation of probiotics associated with prebiotics for vaginal use while probiotics encapsulation is a process widely used in food applications in order to protect microorganisms against stomach acidity (Champagne and Fustier, 2007). One of the most common method used for probiotic encapsulation is based on the gelation of alginate in contact with calcium ions (Krasaekoopt et al., 2003). The main objective of this study was to encapsulate probiotic strains and prebiotic into bioadhesive pectinate-hyaluronic acid microparticles dedicated to the formulation of a vaginal delivery system. In this study, the encapsulation system could enhance the effects of Lactobacillus sp. and protect them during drying process and storage. As alginate, low methyl pectin (LM pectin) forms a gel in contact with divalent cations such as Ca2+ ions. It is generally accepted that the gelation mechanism of LM pectin relies mainly on the well-known ‘‘egg-box model’’ (Grant et al., 1973). However, some authors (Racape et al., 1989) criticized this model for gelation of amidated pectins. The gel formation of LM pectin depends on several factors: pH, nature and concentration of the cross-linking agent (Ca2+, Zn2+. . .), concentration and composition of LM pectin, presence or not of sugar (Löfgren et al., 2006; Bourgeois et al., 2006; Assifaoui et al., 2010). We chose to associate calcium-pectinate with hyaluronic acid sodium salt (HA) in order to combine their bioadhesive properties suitable for vaginal use (Valenta, 2005; Asane et al., 2008; Rinaudo, 2008). Moreover, the two polymers can repair vaginal dryness. Pectin can also decrease vaginal residue (Caswell and Kane, 2002) and the combination of HA with Döderlein’s bacillus could improve Lactobacillus sp. effectiveness to normalize vaginal ecosystem (Baldacci, 1997; Valenta, 2005). The first step of this study was to prepare unloaded bioadhesive microparticles based on pectinate/HA mixture with a size between 100 and 200 lm. These preliminary experiments allowed to welldefine the experimental factors influencing the microparticle size distribution as well as their experimental range. Then, an experimental design has been realized to improve the operating conditions of the microencapsulation process and obtain bioadhesive microparticles able to encapsulate the pro- and prebiotics.
2. Materials and methods 2.1. Materials Calcium chloride (CaCl2), hyaluronic acid sodium salt (HA), porcine stomach mucins, potassium hydroxide (KOH), calcium hydroxide (Ca(OH)2), bovine serum albumin, lactic acid, acetic acid, glycerol, urea, glucose, EDTA, TweenÒ 80, SpanÒ 80, methylene blue and Eriochrome black T were purchased from Sigma–Aldrich (St. Quentin Fallavier, France). Low methyl pectin was obtained from CargillÒ (St. Germain en Laye, France). Vegetal oil (C8/C10) and stearate sucroesters were obtained from Stéarinerie Dubois (Boulogne-Billancourt, France). Fructo-oligosaccharides (prebiotics), FOS ActilightÒ 950S and 950P were a gift of Béghin Meiji (Marckolsheim, France). Four selected commercial probiotic strains including Lactobacillus rhamnosus, L. salivarius, L. brevis and L. plantarum were a gift of NebraskaÒ cultures (Walnut Creek, California, USA). The probiotic organisms were maintained individually at 20 °C. The Man Rogosa Sharpe (MRS) broth was purchased from BD DifcoTM (Pont de Claix, France). Acridine orange was purchased from Alfa Aesar GmbH & Co KG (Karlsruhe, Germany). 2.2. Microencapsulation process The microencapsulation process was performed according to an emulsification/gelation method (Capela et al., 2007; Sheu et al., 1993). Briefly, 37.5 ml of an aqueous solution made of sterile LM pectin, hyaluronic acid sodium salt (HA) and fructooligosaccharides (FOS) was prepared in presence or not of probiotics. Different polymer and FOS concentrations were studied: from 2% to 7% (w/v) for LM pectin, from 0.5% to 1% (w/v) for HA and from 2% to 10% (w/ v) for FOS. This aqueous solution was then emulsified into a vegetal oil containing surfactant (Polysorbate 80 or stearate sucroester) for 30 min. Different impeller types at 1200 rpm were evaluated as stirring device as well as an ultra-turraxÒ at 13,500 rpm. Then, 150 ml of calcium chloride (0.1 M or 0.2 M) were added under stirring (with an addition rate ranging from 10 to 20 ml/min) inducing the formation of calcium pectin/HA microparticles. The addition of an aqueous solution containing 0.9% (w/v) of NaCl allowed the emulsion breakage. Then, the oil layer was drained, the microparticles were removed from the aqueous phase by filtration and washed twice with saline water. Microparticles were stored at +4 °C into physiological water. 2.3. Microparticle characterization 2.3.1. Size The size distribution of pectin/HA/Ca2+ microparticles were measured by laser light scattering (Mastersizer 2000, Malvern Instruments, UK). 2.3.2. Microparticle morphology Microparticles were observed by optical microscopy after adding methylene blue inside the loaded microparticle suspension. A blue coloration of probiotic loaded-microparticles was observed while unloaded microparticles remained uncolored. This technique was also very helpful to quickly verify probiotic encapsulation. 2.4. Complexometric determination of Ca2+ ions amount into pectin/ HA beads Ca2+ ions entrapped into microparticles were quantified by ethylene diaminetetraacetic acid (EDTA) titration. EDTA is a chelating agent complexing with most metal ions. The equivalence point is
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detected through the use of the indicator Eriochrome Black T which is itself a chelating agent. After microparticle formulation and oil removal, the aqueous phase was isolated. 10 ml of the aqueous phase and 10 ml basic equimolar buffer with a pH = 9.50 (ammonium hydroxide, chloride hydroxide) were introduced into Erlenmeyer flask. Before titration some droplets of an ethanol solution of Eriochrome Black T were added and the aqueous phase was titrated by EDTA (0.1 M). 2.5. Bioadhesion evaluation by rheological measurements Rheological measurements were conducted on pectinate or pectinate/HA gels in order to evaluate their bioadhesive properties. Pectin or pectin/HA solutions were prepared by dispersing the required amounts of polymers in 0.9% (w/v) NaCl. All samples were stirred at +80 °C for 1 h. About 5% (w/v) of FOS were added to each sample. The pH of all solutions was set to 4.2. Then, pre-heated solution of CaCl2 was added in the polymer solutions under stirring during 1 min. The gels obtained were stored at room temperature during 48 h before rheological measurements. Rheological measurements were carried out at +37 °C using a Bohlin VOR controlled rate rheometer using a parallel plate (PP30) measuring system. After the identification of the linear viscoelastic region at 1 Hz, gels were investigated over a frequency range from 0.03 to 5 Hz. All samples were prepared in duplicate and each of them was analyzed five times. 2.6. Probiotic characterization 2.6.1. Evaluation of probiotic bacteria encapsulation using an acridine orange fluorescence labeling This technique consisted in labeling probiotics with acridine orange, which is a nucleic acid selective fluorescent cationic dye useful for cell cycle determination. Acridine orange which is excited under blue light, has the faculty to be cell-permeable and to interact with DNA by intercalation and RNA by electrostatic interactions. DNA intercalated acridine orange fluoresces at yellow-green wavelength (525 nm) while RNA electrostatically bounded acridine orange fluoresces at red ones (>630 nm). Before the encapsulation process, an acridine orange solution was added to the probiotic suspension under magnetic stirring during 10 min. Then probiotics were isolated by filtration, washed twice and introduced in the polymer solution. Once microparticles were formed, the suspension was observed by confocal microscopy. Acridine orange fluorescence technique is an easy and sensitive method for screening biological samples. However in some case it could be difficult to well-distinguish between yellow-green and red colors. Moreover the contact time and the concentration of acridine orange could spark off these color variations (Maki and Remsen, 1981) and the colors observed not always translate the bacteria activity (Quinn, 1984). Therefore this technique has been completed by the pour plate technique using MRS agar so as to confirm or not the viability of probiotic strains. 2.6.2. Enumeration of probiotic bacteria To determine the viable counts of the encapsulated probiotic bacteria, microparticles were dispersed in phosphate buffer. After complete release of probiotics, serial dilutions were prepared with peptone water and culture was plated by the pour plate technique using MRS agar. Plates were incubated in jars Anaerobac anaerobiosis-generating systems at +37 °C for 48 h. Platting was carried out in triplicate. 2.6.3. Kinetic release of probiotics from microparticles The release profile of probiotics from microparticles of the improved formulation was followed during 24 h. The in vitro release
medium was a simulated vaginal fluid (SVF) at pH 4.2 whose composition is described in Table 1 (Owen and Katz, 1999; Das Neves et al., 2008). About 0.100 g of microparticles were dispersed under magnetic stirring at +37 °C in 9 ml of SVF since daily vaginal secretions were estimated in a range of 1–11 ml (Owen and Katz, 1999) After incubation time of 30 min, 1, 3, 5, 7, 16 and 24 h, all samples were filtered in order to only recuperate a bacterial suspension. The enumeration of the released probiotic bacteria was then performed by the pour plate technique using MRS agar as described previously. Plates were incubated in Anaerobac jars (anaerobiosis-generating systems) at +37 °C for 48 h. Platting was carried out in duplicate. 3. Results and discussion 3.1. Preliminary experiments The influence of several formulation and process parameters (Table 2) was first evaluated on the size distribution of unloaded microparticles. 3.1.1. Process parameters From a technical point of view, the choice of an impeller type depends on the direction (radial or axial) of the flow to be generated. The encapsulation process was made of two very different steps: (i) an emulsion step for which shear forces (radial dispersion) are needed in order to produce relatively monodisperse and small droplets and (ii) a gelation step requiring axial flow to mix efficiently the added Ca2+ solution with pectin gel. Therefore, three impeller types were evaluated to prepare the microparticles: a four-blade impeller inducing axial dispersion, a Rushton turbine and an ultra-turraxÒ homogenizer suitable to generate high shear forces. Similar size distributions (with a mean size around 200 lm) were measured for microparticles prepared by using a four blade impeller or a Rushton turbine (results not shown). As expected, the use of an ultra-turraxÒ homogenizer at 13,500 rpm during the emulsification step allowed to obtain smaller microparticles with a mean size comprised between 20 and 60 lm. This size range made the removal of microparticles from the oil phase much more difficult with only a small portion of them being recovered. Moreover homogenization using ultra-turraxÒ led to local heating of the emulsion that could alter probiotic viability. From an energetical point of view, the four-blade impeller was the best stirring system able to produce satisfying microparticles and to preserve probiotics during the encapsulation process. 3.1.2. Oil type and polymer:oil ratio The oil type was found to play an important role to avoid aggregate formation during the gelation step. Different oil types have been studied: mineral oils (paraffin, cyclopentasiloxan, pentan) and vegetal oils (sunflower, triglyceride mixtures). Oil viscosity
Table 1 Simulated vaginal fluid composition. Component quantity (g/l) Porcine gastric mucin (type II) NaCl KOH Ca(OH)2 Bovine serum albumin Lactic acid Acetic acid Glycerol Urea Glucose
15 3.51 1.40 0.222 0.018 2.00 1.00 0.16 0.4 5.0
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Table 4 HLB of surfactant used.
Formulation and process parameters HLB
Oil type (mineral and vegetal oil) Polymer:oil ratio [Polymers] (HA and LM pectin) Impeller type [Prebiotics] Type of surfactant [Surfactant]
reported in Table 3 had a significant effect on the size distribution and the morphology of the microparticles. Low viscosity oils (0– 10 mPa s) led to very large microparticles with a mean diameter of around 4 mm and more. For oil viscosity higher than 50 mPa s, as for sunflower and paraffin oils, the mean diameter of the microparticles ranged from 200 up to 700 lm. In this case, the formation of aggregates was observed, microparticles filtration became uneasy and the oil excess was difficult to remove. The triglyceride (TG) mixture allowed a good compromise between the formulated microparticle size and their ease of use for the entire formulation process. Indeed regular microparticles were obtained without any aggregate formation during the calcium addition. Elimination of the oil phase was easier and only a few oily residues remained in the final microparticle suspension. Moreover, no significant difference on the particle mean diameter was observed by varying the C18/C10 composition of the triglyceride mixture (70/20; 65/35; 55/45). A polymer:oil ratio of 1:4 was selected so as to obtain a good dispersion of the polymer solution inside the oil phase and to facilitate the CaCl2 solution addition. For larger polymer amounts (corresponding to polymer:oil ratios of 1:2 and more), the emulsification of the polymer solution became more difficult as well as the addition of calcium solution leading to the formation of microparticle aggregates. 3.1.3. Concentration and type of surfactants Once the oil phase defined, polymer in triglycerides emulsions have been formulated by using different surfactants in order to estimate the required HLB of the triglyceride mixture. Two surfactant couples were studied: TweenÒ 80/SpanÒ 80 and two sucroesters (5S and 15S) whose HLB are reported in Table 4. For each surfactant couple, the studied HLB range was the following: 5, 5.5, 6, 6.5, 7, 8, 9, 10 and 15. In each case, W/O emulsions were obtained. Coalescence between droplets was observed by optical microscopy for emulsions stabilized by TweenÒ 80/SpanÒ 80 couple while sucroesters led to stable emulsions. The required HLB of the TG mixture was 5 corresponding to the sucroester 5S HLB. In the classical emulsification/gelation process (Sheu et al., 1993), TweenÒ 80 surfactant is used to facilitate the removal of the oil phase at the end of the process for an easier microparticle separation. We decided to replace this hydrophilic surfactant by the sucroester 5S to preferentially stabilize the W/O emulsion. The microparticles exhibited monomodal size distribution which were quite similar with a mean diameter about 110 lm for TweenÒ
Table 3 Viscosity of each oil type measured at +25 °C. Oil type
Viscosity (mPa s)
Pentan Cyclopentasiloxan Triglyceride mix Sunflower Paraffin
0.225 4 28–3158 58 170
TweenÒ 80
Span 80
Sucroester 5S
Sucroester 15S
15
4.3
5
15
80 formulation and 99 lm for sucroester 5S ones (results not shown). The type of surfactant did not significantly influence the particle size distribution but had an impact on microparticle shape. The use of sucroester 5S inside the oil phase led to the formation of stable emulsions and spherical and regular microparticles while microparticle aggregates were observed with TweenÒ 80. We assumed that the emulsion stabilized with 5S sucroester did not immediately break with the CaCl2 solution addition as in the case of TweenÒ 80 emulsion. 3.1.4. Polymer concentration The bioadhesivity of different pectin gels prepared with or without HA was evaluated by a rheological method based on the comparison of their storage moduli. These measurements were generally realized in presence or not of mucins so as to estimate the interactions between polymer gels and mucin. The interaction parameter DG0 is calculated from the equation:
G0mix ¼ G0p þ G0m þ DG0 G0mix ,
G0p
ð1Þ
G0m
where and are the elastic (storage) moduli of the mixture, the polymer and the mucins, respectively. The absolute synergism parameter DG0 is the elastic component which translates the interaction between polymers and mucin (Rossi et al., 1995; Hägerström et al., 2000; Andrews and Jones, 2006). The elastic modulus of the mucin dispersion being negligible, the following simplified equation is generally used to calculate DG0 :
G0mix ¼ G0p þ DG0
ð2Þ
Moreover, in the case of the calcium-pectinate gels, mucins could interact with Ca2+ ions reducing the interaction between pectin and Ca2+ ions that led to weakly gels. Indeed, divalent cations could be bound by the negative charges of mucus sialic acid (Park and Robinson, 1985). In particular, Hägerström et al. (2000) have shown that mucin addition could lead to the destruction of gel structure. Therefore we decided not to use mucins in our study assuming that an increase of the storage modulus (i.e. DG0 > 0) translates an increase of the adhesion effect. Different concentrations of polymers were used to prepare calcium-pectinate gels: LM pectin concentration ranged from 2% up to 7% (w/v) and HA from 0.5% to 1% (w/v). As expected, the obtained results for the pectin/Ca2+ gels (Fig. 1) showed an increase of the storage modulus G0 with the pectin concentration. The size distributions of microparticles prepared with 3%, 5% or 7% (w/v) pectin are presented in Fig. 2. A monomodal distribution was observed for 3% (w/v) of pectin with a mean diameter of 102 lm. Higher pectin concentrations led to more viscous solutions decreasing the stirring efficiency and inducing multimodal size distributions with higher mean diameters of 128 and 303 lm for 5% and 7% (w/v) pectin, respectively. Then, rheological measurements have been performed on different gels of pectin/Ca2+ and pectin/Ca2+/HA in order to evaluate the influence of HA addition on adhesive properties of pectin gels. HA was added at 0.5% or 1% (w/v). The evolution of G0 modulus presented in Fig. 1 shows that HA addition inside pectin/Ca2+ gels could enhance the gel adhesive properties. Higher adhesion effect was observed for pectin/Ca2+/0.5% (w/v) HA gels whatever pectin concentration. For 1% (w/v) of HA, the conclusions were not so clear. An improvement of the adhesion effect was clearly observed for gel with at least 5% (w/v) of pectin. For 4% pectin, no significant
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Fig. 1. Evaluation of the adhesive properties of (j) LM pectin/Ca2+ gels, (4) LM pectin/Ca2+/HA 0.5% (w/v) gels and (s) LM pectin/Ca2+/HA 1% (w/v) gels by measuring the evolution of G’ as a function of pectin concentration.
Fig. 2. Influence of pectin concentration on microparticle size distribution: (dash line) pectin 3% (w/v), (dotted line) pectin 5% (w/v) and (dotted dash line) pectin 7% (w/v).
evolution of G0 was observed and for lower pectin concentrations, the addition of HA at 1% (w/v) induced a decrease of G0 indicating the formation of weaker gels. Calcium diffusion in an alginate solution leads to gelation by complexing with the polymer carboxylic groups which is translated by a significant loss of water against the current of Ca2+ flux, during the gelation process (Yotsuyanagi et al., 1987). A study on alginate–hyaluronate gel and beads (Oerther et al., 1999) investigated the gel formation between alginate, HA and Ca2+ ions showing that HA addition reduced the dehydration effect of gels and that interactions between HA and Ca2+ ions could be dependent on the composition of the polymer mixtures. HA addition could disturb the diffusion of Ca2+ ions and also the equilibrium of water and Ca2+ ions content in the gel. In this way HA addition leads to a modification of gel structure and reduced hardness of gel. Furthermore Ca2+ ions diffusion in alginate gel (without HA) appeared slower than in gels containing a little amount of HA. They recommended to use a high molecular weight and a moderate concentration of HA so as to make gel easier. To our knowledge, no study evaluated interaction between HA and pectin, but as alginate presents the same properties as pectin with Ca2+ ions, similar phenomena inside pectin/Ca2+/HA gels could be suggested. In our study, the gels obtained with 0.5% (w/v) HA presented stronger structure than with 1% (w/v) HA according to the G0 values (Fig. 1). We could conclude that addition of 0.5% (w/v) HA had no
real impact on pectin gelation process. At 1% (w/v) HA and especially for low pectin concentration, we could expect that Ca2+ diffusion was significantly limited leading to important hydration effect and a weaker gel structure. The gels could get stronger structure with an increase of pectin concentration until counterbalancing the HA effect for pectin concentrations above 4% (w/v). These observations confirm the results of Oerther et al. (1999). To obtain efficient pectin gelation and adhesion effect at low pectin concentration, HA concentration was set at 0.5% (w/v) for the following experiments, especially for the experimental design. The addition of HA in pectin gels used for the preparation of microparticles did not significantly influence the particle size distribution (Fig. 3). However, larger particles have been obtained with HA addition (102 lm for 0% (w/v) HA, 156 lm for 0.5% (w/ v) HA, 136 lm for 1% (w/v) HA) probably due to the increase of the dispersed phase viscosity.
3.1.5. Prebiotics concentrations Prebiotics are known as nutrients which stimulate the Lactobacilli growth to the detriment of pathogens (Lee and Salminen, 2009). Prebiotics were also added inside microparticles in order to complete the probiotic effect. The influence of prebiotics concentration in pectin gels on microparticle size distribution was investigated.
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Fig. 3. Influence of HA concentration on microparticle size distribution: (dotted line) pectin 3% (w/v), (dotted dash line) pectin 3% (w/v)/HA 0.5% (w/v) and (dash line) pectin 3% (w/v)/HA 1% (w/v).
A
B
Fig. 4. Prebiotic influence on microparticle size distribution with : (A) pectin 3% (w/v)/HA 1% (w/v) and FOS : (continuous line) 0%, (dotted line) 2% (w/v), (dash line) 5% (w/v), (dotted dash line) 10% (w/v) ; (B) pectin 3% (w/v) with 5% (w/v) FOS and (continuous line) HA 1% (w/v) or (dash line) HA 0.5% (w/v).
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3.2. Experimental design
Table 5 Determination of Ca2+ ions amount inside microparticles. Microparticles composition
Ca2+ mole ion number (for 1 g of LM pectin) (mmol)
3% (w/v) of Pectin [CaCl2] = 0.1 M 3% (w/v) of Pectin + 5% (w/v) of FOS [CaCl2] = 0.1 M 3% (w/v) of Pectin + 5% (w/v) of FOS + 0.5% HA [CaCl2] = 0.1 M 3% (w/v) of Pectin + 5% (w/v) of FOS + 0.5% HA [CaCl2] = 0.2 M
3.7 ± 0.1 3.8 ± 0.1 2.9 ± 0.1 5.4 ± 0.1
Table 6 Set parameters for experimental design. Set parameter Oil type Polymer:oil ratio [HA] [Prebiotics] Impeller type Surfactant
Triglyceride mix (C8/C10) 1:4 0.5% (w/v) 5% (w/v) Four blade impeller Sucroesters 5S (HLB = 5)
The size distribution of microparticles made of 3% (w/v) pectin and 1% (w/v) HA in presence or not of prebiotics were first measured (Fig. 4A). Prebiotic concentration had a significant impact on the particle size distribution with a mean diameter of 261 lm for 2% (w/v) prebiotics, 177 lm for 5% (w/v) prebiotics and 199 lm for 10% (w/v) prebiotics. The addition of prebiotics increased the microparticle size especially for 2% and 10% prebiotics. According to our previous results, prebiotics could have an impact on HA faculty to reduce gel dehydration promoting microparticle swelling. We did not find any explanations describing the real impact of prebiotics on the microparticle size distribution. Fu and Rao (2001) have studied sucrose effect on low methyl pectin gelation. They observed that sucrose had the faculty to reduce the water content promoting polymer–polymer interactions rather than polymer–solvent interactions. Furthermore, sucrose could stabilize the cross-linking junction in gel thanks to additional hydroxyl groups that promoted hydrogen bonds to immobilize free water. HA concentration being set at 0.5% (w/v) for mucoadhesion reasons, we decided to compare the size distribution of microparticles obtained with 5% (w/v) prebiotics and 1% or 0.5% (w/v) of HA. No significant difference on size distribution was observed (Fig. 4B). Therefore this prebiotic concentration was chosen for further experiments. 3.1.6. Complexometric determination of Ca2+ ions into microparticles The quantification of Ca2+ involved in the pectin/HA mixture gelation was performed for some formulations. The results obtained are reported in Table 5 and showed that prebiotic addition did not significantly affect the LM pectin gelation. On the contrary, HA addition led to a decrease of Ca2+ ions inside the microparticles suggesting, as discuss above, that HA presence could slow down Ca2+ ions diffusion and therefore the gelation process. As expected, the use of a more concentrated CaCl2 solution, allowed the penetration inside the microparticles of a larger content of calcium ions.
The preliminary experiments allowed the setting of several parameters which are reported in Table 6: the type of oil and surfactant, the oil/polymer ratio, HA and prebiotics concentrations and the impeller type. We also well-defined the experimental range of the remaining operating parameters (Table 7) whose effect on microparticle properties (size and encapsulation efficiency) was investigated by the means of an experimental design. For all these experiments, probiotics strains were added to the polymer solution contrary to the preliminary experiments. Considering the great number of experimental factors (7 parameters), a two-levels fractional factorial design was used for screening purpose allowing the identification of 16 runs (Table 8). This experimental design was built by aliasing the main effects of the factors studied with high-order interactions assumed to be negligible. The chosen factors with their coded levels 1, 0 and + 1 are summarized in Table 7. The measured responses were the microparticle mean diameter d [0.5] and the probiotic encapsulation rate (Table 8). The use of screening design consists in approximating the responses to be measured (Y) by a first-order model whose coefficients bi correspond to the main effects of the experimental factors (Xi) to be studied (Box et al.,2005):
Y ¼ b0 þ
7 X
bi X i
ð3Þ
i¼1
The coefficients bi were calculated by least squares linear regression (Fig. 5) and the results were analyzed by NEMRODWÒ software version 2000 (Nemrodw, LPRAI, Marseille, France). The constant b0 corresponds to the average of the responses for the 16 runs. The factors strongly influencing the response are those having the highest absolute value for bi. The sign of the coefficient shows how the factor influences the response: if the coefficient is negative, the response decreases when the factor ranges from the coded level (1) to (+1); the contrary is obtained if the coefficient is positive. Then, coefficient signs allow the determination of the best coded level for each factor. The coefficient calculation (Fig. 5) highlighted two significant factors influencing the microparticle mean size which varied from 87 to 729 lm depending on the operating conditions applied: the stirring rate (X1) and the pectin concentration (X3). The stirring rate (X1) had to be increased at the higher level (1200 rpm) so that the particle mean size decreased below 380 lm. An increase in pectin quantity (X3) led to an increase in particle mean size. This effect could be attributed to a reduction of stirring efficiency induced by the increase of the organic phase viscosity. Moreover, bimodal distributions were obtained for microparticles made with 5% (w/ v) of pectin while monomodal distributions characterized the formulations based on 3% (w/v) of pectin (Fig. 6). The pectin concentration at 3% (w/v) was then preferred. By considering the standard deviation about 15 lm of the D (0.5) estimated through the replication of experiments, results surprisingly showed no significant effect of the surfactant concentration (X5) on the particle size for the experimental range studied.
Table 7 Design factors and their levels.
Levels 1 0 +1
Stirring rate (rpm)
Emulsification time (min)
Pectin (%)
CaCl2 (M)
Surfactant (%)
Gelation time (min)
Addition rate of CaCl2 (ml/min)
X1 720 960 1200
X2 10 20 30
X3 3 4 5
X4 0.1 0.15 0.2
X5 0.02 0.11 0.2
X6 10 15 20
X7 10 15 20
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Table 8 Experimental design. No. exp.
Stirring rate (rpm)
Emul. time (min)
[Pectin] (%(w/v))
[CaCl2] (M)
[Surfactant] (%(w/v))
Gel. time (min)
Addition rate of CaCl2 (ml/min)
D (0.5) (lm)
Encapsulation rate (108 CFU/g microparticles)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
720 1200 720 1200 720 1200 720 1200 720 1200 720 1200 720 1200 720 1200
10 10 30 30 10 10 30 30 10 10 30 30 10 10 30 30
3 3 3 3 5 5 5 5 3 3 3 3 5 5 5 5
0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2
0.02 0.2 0.2 0.02 0.02 0.2 0.2 0.02 0.2 0.02 0.02 0.2 0.2 0.02 0.02 0.2
10 20 20 10 20 10 10 20 10 20 20 10 20 10 10 20
10 20 10 20 20 10 20 10 20 10 20 10 10 20 10 20
229 87 132 137 640 357 729 195 453 171 377 104 686 379 539 239
1.53 1.43 1.38 1.53 1.42 1.56 1.44 1.58 1.74 2.41 1.37 1.59 1.51 1.69 1.96 1.72
Fig. 5. The main effects of the experimental factors on the particle mean diameter.
Fig. 7. Methylene blue labeled microparticles obtained with pectin 3% (w/v), HA 0.5% (w/v) and FOS 5% (w/v) (experiment no. 4) observed by optical microscopy (40). Fig. 6. Pectin concentration influence on the size distribution of probiotic-loaded microparticles obtained from experiment no. 4 (dotted line) with pectin 3% (w/v) or from experiment no. 14 (dotted dash line) with pectin 5% (w/v).
The other factors had reduced influence compared to the stirring rate and the pectin concentration. The measured values for the second response, the probiotic encapsulation rate, varied between 1.4 108 and 2.4 108 CFU/g
of microparticles. These results were considered quite similar according to the experimental error estimated by the replication of experiments with a standard deviation about 0.6 108 CFU/g of microparticles. This indicates that the operating factors studied did not affect significantly the probiotic encapsulation rate in the experimental range studied.
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Fig. 8. Acridine orange labeled microparticles obtained with pectin 3% (w/v), HA 0.5% (w/v) and FOS 5% (w/v) (experiment no. 4) observed by (A) transmission and (B) confocal microscopy (20).
Fig. 9. Percentage of probiotics released in vitro from microparticles obtained with pectin 3% (w/v), HA 0.5% (w/v) and FOS 5% (w/v) (experiment no. 4) as a function of time.
The operating conditions for experiment no. 4 were consistent with the conclusions drawn from the experimental design study and led to interesting properties for our application being a good compromise between the mean diameter (137 lm) and the size distribution of the microparticles. The reproducibility of this formulation was then verified. Fig. 7 represents a microscopic observation of the improved formulation containing methylene blue showing an effective encapsulation of the probiotics. Indeed for probiotic-loaded microparticles, methylene blue remained fixed on probiotics that led to a blue coloration of microparticles. For unloaded microparticles no coloration was observed. Moreover, acridine orange fluorescence was used to specifically label Lactobacillus sp. (as described in Section 2). This labeling allowed confirming that probiotic strains were inside the microparticles and not in the external phase of the suspension (Fig. 8). Finally, the release profile of probiotics from microparticles of the improved formulation (experiment no. 4) was evaluated. Fig. 9 shows a sustained release of the probiotics during 10 h.
The probiotics were totally released after 16 h incubation and a stabilization of the bacteria population was observed between 16 and 24 h. The percentage values obtained exceeding 100 could reflect the beginning of the probiotic proliferation. This type of release profile is interesting for a vaginal application since microparticles allowed a sustained release of probiotics followed by a bacteria proliferation. This aspect will be more investigated from the final form (vaginal tablets incorporating-microparticles) in a future work. 4. Conclusion Pro-and pre-biotics-loaded bioadhesive microparticles made of HA and low methyl pectin have successfully been developed for a pharmaceutical use. The operating conditions allowing the preparation of favorable microparticles for probiotic encapsulation have been determined through an experimental design. The microparticle obtained exhibited adhesive properties as shown by the rheological measurements and a sustained release of probiotics. In
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the future, incorporation of these microparticles into vaginal tablet will be evaluated. Then, the optimization of the tablet composition and the ‘‘tablet’’ process will be performed in relation with the viability, the encapsulation rate and the delivery kinetics in the vaginal tract of the probiotic strains. Moreover bioadhesion properties of gels, microparticles and tablets will be investigated. References Ahuja, A., Khar, R.K., Ali, J., 1997. Mucoadhesive drug delivery system. Drug Dev. Ind. Pharm. 23 (5), 489–515. Alambar, H., Akhrul, A., 2005. The vagina as a route for systemic drug delivery. J. Control. Release 103, 301–313. Andrews, G.P., Jones, D.S., 2006. Rheological characterization of bioadhesive binary polymeric systems designed as platforms for drug delivery implants. Biomacromolecules 7, 899–906. Asane, G.S., Nirmal, S.A., Rasal, K.B., Naik, A.A., Mahadik, M.S., Rao, Y.M., 2008. Polymers for mucoadhesive drug delivery system: a current status. Drug Dev. Ind. Pharm. 34, 1246–1266. Assifaoui, A., Loupiac, C., Chambin, O., Cayot, P., 2010. Structure of calcium and zinc pectinate films investigated by FTIR spectroscopy. Carbohydrate Res. 345 (7), 929–933. Baldacci, M., 1997. Pharmaceutical compositions for topical use containing hyaluronic acid and doederlein’s bacillus for the prevention and treatment of vaginal diseases. EP 769298. Baloglu, E., Senyigit, Z.A., Karavana, S.Y., Bernkop-Schnürch, A., 2009. Strategies to prolong the intravaginal residence time of drug delivery systems. J. Pharm. Pharm. Sci. 12, 312–336. Bourgeois, S., Gernet, M., Pradeau, D., Andremont, A., Fattal, E., 2006. Evaluation of critical formulation parameters influencing the bioactivity of beta-lactamases entrapped in pectin beads. Int. J. Pharm. 324 (1), 2–9. Box, G.E., Hunter, W.G., Hunter, J.S. Statistics for Experimenters, second ed. John Wiley, New Jersey, 2005. Capela, P., Hay, T.K.C., Shah, N.P., 2007. Effect of homogenization on bead size and survival of encapsulated probiotic bacteria. Food Res. Int. 40, 1261–1269. Caswell, M., Kane, M., 2002. Comparison of the moisturization efficacy of two vaginal moisturizers: pectin versus polycarbophil technologies. J. Cosmet. Sci. 53, 81–87. Champagne, C.P., Fustier, P., 2007. Microencapsulation for the improved delivery of bioactive compounds into foods. Curr. Opin. Biotechnol. 18, 184–190. Das Neves, J., Amaral, M.H., Bahia, M.F., 2008. Performance of an in vitro mucoadhesion testing method for vaginal semisolids: influence of different testing conditions and instrumental parameters. Eur. J. Pharm. Biopharm. 69, 622–632. Duchene, D., Touchard, F., Peppas, N.A., 1988. Pharmaceutical and medical aspects of bioadhesive systems for drug administration. Drug Dev. Ind. Pharm. 14, 283– 318. Edsman, K., Hägerström, H., 2005. Pharmaceutical applications of mucoadhesion for the non-oral routes. J. Pharm. Pharmacol. 57, 3–22. FAO/WHO, 2001. Health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. Report of a joint FAO/WHO expert consultation. Available from:. Fu, J., Rao, M., 2001. Rheology and structure development during gelation of lowmethoxyl pectin gels: the effect of sucrose. Food Hydrocolloids 15, 93–100. Gavini, E., Sanna, V., Juliano, C., Bonferoni, M.C., Giunchedi, P., 2002. Mucoadhesive vaginal tablets as veterinary delivery system for the controlled release of an antimicrobial drug, acriflavine. AAPS PharmSciTech 3 (3), E20. Grant, G., Morris, E., Rees, D., Smith, P., Thom, D., 1973. Biological interactions between polysaccharides and divalent cations: the egg-box model. FEBS Lett. 32 (1), 195–198. Gu, J.M., Robinson, J.R., Leung, S.H., 1988. Binding of acrylic polymers to mucin/ epithelial surfaces: structure–property relationships. Crit. Rev. Ther. Drug Carrier Syst. 5, 21–67. Gurny, R., Meyer, J.M., Peppas, N.A., 1984. Bioadhesive intraoral release systems: design, testing and analysis. Biomaterials 5, 336–340. Hussain, A., Ahsan, F., 2005. The vagina as a route for systemic drug delivery. J. Control. Release 103, 301–313. Hägerström, H., Paulsson, M., Edsman, K., 2000. Evaluation of mucoadhesion for two polyelectrolyte gels in simulated physiological conditions using a rheological method. Eur. J. Pharm. Sci. 9, 301–309.
Khutoryanskiy, V.V., 2010. Advances in mucoadhesion and mucoadhesive polymers. Macromol Biosci. Article first published online: 27 DEC 2010. Krasaekoopt, W., Bhandari, B., Deeth, H., 2003. Evaluation of encapsulation techniques of probiotics for yoghurt. Int. Dairy J. 13, 3–13. Lee, Y.K., Salminen, S. (Eds.), Handbook of probiotics and prebiotics. second ed. John Wiley & Sons, Inc., Hoboken, New Jersey, 2009. Lenoir-Wijnkoop, I., Sanders, M.E., Cabana, M.D., Caglar, E., Corthier, G., Rayes, N., Sherman, P.M., Timmerman, H.M., Vaneechoutte, M., Van Loo, J., Wolvers, D.A.W., 2007. Probiotic and prebiotic influence beyond the intestinal tract. Nutr. Rev. 65, 469–489. Löfgren, C., Guillotin, S., Hermansson, A., 2006. Microstructure and kinetic rheological behavior of amidated and non-amidated lm pectin gels. Biomacromolecules 7, 114–121. Macfarlane, S., Macfarlane, G.T., Cummings, J.H., 2006. Review article: prebiotics in the gastrointestinal tract. Aliment. Pharmacol. Ther. 24, 701–714. Maki, J.S., Remsen, C.C., 1981. Comparison of two direct-count methods for determining metabolizing bacteria in freshwater. Appl. Environ. Microbiol. 41 (5), 1132–1138. Oerther, S., Le Gall, H., Payan, E., Lapicque, F., Presle, N., Hubert, P., Dexheimer, J., Netter, P., 1999. Hyaluronate-alginate gel as a novel biomaterial: mechanical properties and formation mechanism. Biotechnol. Bioeng. 63, 206–215. Owen, D.H., Katz, D.F., 1999. A vaginal fluid simulant. Contraception 59, 91–95. Park, H., Robinson, J., 1985. Physico-chemical properties of water insoluble polymers important to mucin epithelial adhesion. J. Control. Release 2, 47–57. Pavelic´, Z., Skalko-Basnet, N., Filipovic´-Grcic´, J., Martinac, A., Jalsenjak, I., 2005. Development and in vitro evaluation of a liposomal vaginal delivery system for acyclovir. J. Control. Release 106, 34–43. Peppas, N., Buri, P., 1985. Surface, interfacial and molecular aspects of polymer bioadhésion on soft tissues. J. Control. Release 2, 257–275. Peppas, N.A., Sahlin, J.J., 1996. Hydrogels as mucoadhesive and bioadhesive materials: a review. Biomaterials 17, 1553–1561. Quinn, J.P., 1984. The modification and evaluation of some cytochemical techniques for the enumeration of metabolically active heterotroph bacteria in the aquatic environment. J.A.B. 57, 51–57. Racape, E., Thibault, J.F., Reitsma, J.C.E., Pilnik, W., 1989. Properties of amidated pectin. ii: polyelectrolyte behaviour and calcium binding of amidated pectins and amidated pectic acid. Biopolymers 28, 1435–1439. Reid, G., 2001. Probiotic agents to protect the urogenital tract against infection. Am. J. Clin. Nutr. 73, 437S–443S. Reid, G., Bruce, A.W., 2003. Urogenital infections in women: can probiotics help? Postgrad. Med. J. 79, 428–432. Richardson, J.L., Illum, L., Thomas, N.W., 1992. Vaginal absorption of insulin in the rat: effect of penetration enhancers on insulin uptake and mucosal histology. Pharm. Res. 9, 878–883. Richardson, J.L., Ramires, P.A., Miglietta, M.R., Rochira, M., Bacelle, L., Callegaro, L., Benedetti, L., 1995. Novel vaginal delivery systems for calcitonin: i: evaluation of hyaff/calcitonin microspheres in rat. Int. J. Pharm. 115, 9–15. Richardson, J., Ramirez, A., Miglietta, M.R.E.A., 1996. Novel vaginal delivery systems for calcitonin: ii. preparation and characterization of hyaff microspheres containing calcitonin. Int. J. Pharm. 144, 19–26. Rinaudo, M., 2008. Main properties and current applications of some polysaccharides as biomaterials. Polym. Int. 57, 397–430. Rochira, M., Miglietta, M.R., Richardson, J.L., Ferrari, L., Beccaro, M., Benedetti, L., 1996. Novel vaginal delivery systems for calcitonin ii: preparation and characterization of hyaff microspheres containing calcitonin. Int. J. Pharm. 144, 19–26. Rossi, S., Bonferoni, M.C., Lippoli, G., Bertoni, M., Ferrari, F., Caramella, C., Conte, U., 1995. Influence of mucin type on polymer–mucin rheological interactions. Biomaterials 16, 1073–1079. Rousseau, V., Lepargneur, J.P., Roques, C., Remaud-Simeon, M., Paul, F., 2005. Prebiotic effects of oligosaccharides on selected vaginal Lactobacilli and pathogenic microorganisms. Anaerobe 11, 145–153. Sheu, T.Y., Marshall, R.T., Heymann, H., 1993. Improving survival of culture bacteria in frozen desserts by microentrapment. J. Dairy Sci. 76, 1902–1907. Smart, J.D., 2005. The basics and underlying mechanisms of mucoadhesion. Adv. Drug Deliv. Rev. 57, 1556–1568. Valenta, C., 2005. The use of mucoadhesive polymers in vaginal delivery. Adv. Drug Deliv. Rev. 57, 1692–1712. Woodley, J., 2001. Bioadhesion: new possibilities for drug administration? Clin. Pharmacokinet. 40, 77–84. Yotsuyanagi, T., Ohkubo, T., Ohhashi, T., Ikeda, K., 1987. Calcium-induced gelation of alginic acid and ph sensitive reswelling of dried gels. Chem. Pharm. Bull. 35 (4), 1555–1563.
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European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps
Improvement of an encapsulation process for the preparation of pro- and prebiotics-loaded bioadhesive microparticles by using experimental design D. Pliszczak a,b,⇑, S. Bourgeois b,2, C. Bordes a,1, J.P. Valour b,2, M.A. Mazoyer c, A.M. Orecchioni d, E. Nakache e, P. Lantéri a a
Université de Lyon, F-69622, Lyon, France; Université Lyon 1, Villeurbanne; Laboratoires des sciences analytiques (LSA) UMR 5180, CNRS, CPE, 43 bd du 11 novembre 1918, 69100 Villeurbanne, France Université de Lyon, F-69622, Lyon, France; Université Lyon 1, Villeurbanne; Laboratoire d’Automatique et de Génie des Procédés (LAGEP), ISPB, UMR 5007, CNRS, CPE, 43 bd du 11 novembre 1918, 69100 Villeurbanne, France c Département pédagogique des sciences biomédicales A, ISPB faculté de pharmacie, Université Lyon 1, France d UFR de Médecine et Pharmacie, Université de Rouen, 22 boulevard Gambetta, 76000 Rouen, France e LCMT, UMR CNRS 6507, ENSICAEN, 6 Boulevard Maréchal Juin, 14000 CAEN, France b
a r t i c l e
i n f o
Article history: Received 4 April 2011 Received in revised form 15 June 2011 Accepted 19 June 2011 Available online 25 June 2011 Keywords: Vaginal delivery system Pectin Hyaluronic acid Probiotics Prebiotics Bioadhesive microparticles
a b s t r a c t The purpose of this study was to design a new vaginal bioadhesive delivery system based on pectinate– hyaluronic acid microparticles for probiotics and prebiotics encapsulation. Probiotic strains and prebiotic were selected for their abilities to restore vaginal ecosystem. Microparticles were produced by emulsification/gelation method using calcium as cross-linking agent. In the first step, preliminary experiments were conducted to study the influence of the main formulation and process parameters on the size distribution of unloaded microparticles. Rheological measurements were also performed to investigate the bioadhesive properties of the gels used to obtain the final microparticles. Afterwards an experimental design was performed to determine the operating conditions suitable to obtain bioadhesive microparticles containing probiotics and prebiotics. Experimental design allowed us to define two important parameters during the microencapsulation process: the stirring rate during the emulsification step and the pectin concentration. The final microparticles had a mean diameter of 137 lm and allowed a complete release of probiotic strains after 16 h in a simulated vaginal fluid at +37 °C. Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction The vagina is a large cavity which offers a potential route of administration for a local or a systemic drug delivery. Traditionally, solutions, suppositories, tablets, sponges, gels and foams have been used as vaginal formulations. The most common use is for a local delivery, especially for female-related conditions as antimicrobial, antifungal, antiviral, spermicidal agents, anti-inflammatory, prostaglandins and steroids (Hussain and Ahsan, 2005; Baloglu et al., 2009). According to the delivery system used, drug absorption, distribution and residence time in the vaginal tract may be different (Alambar and Akhrul, 2005). For example foams, solutions and suspensions allowed obtaining better coverage of vaginal tissue than ⇑ Corresponding author. Université de Lyon, F-69622, Lyon, France; Université Lyon 1, Villeurbanne; Laboratoires des sciences analytiques (LSA) UMR 5180, CNRS, CPE, 43 bd du 11 novembre 1918, 69100 Villeurbanne, France. Tel: +33 0 472448121/31844. E-mail address: [email protected] (D. Pliszczak). 1 Tel: +33 0 472448121. 2 Tel : +33 0 472431844 0928-0987/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2011.06.011
tablets (Hussain and Ahsan, 2005). Nonetheless liquid formulations are inappropriate for controlled drug release due to their short residence time in the vaginal tract. To obtain a local effect, semi-solid or fast dissolving solid bioadhesive system is required so as to uniformly distribute drug inside the vagina. Vaginal inserts and tablets allowed obtaining a continuous drug release for several hours (Alambar and Akhrul, 2005; Hussain and Ahsan, 2005; Baloglu et al., 2009). Particulate-delivery systems, as for example microspheres and liposomes, have been used for local or systemic action to deliver various drugs such as: acyclovir (Pavelic´ et al., 2005), calcitonin (Richardson et al., 1995, 1996; Rochira et al., 1996), insulin (Richardson et al., 1992), or acriflavine (Gavini et al., 2002). The main problem for vaginal drug delivery is the presence of cervical mucus which could rapidly remove delivery systems. Therefore physical factors of the vaginal tract have to be considered since many variations as cyclic changes, thickness of vaginal mucosa, properties of the vaginal fluid and sexual activity could affect drug delivery across the vaginal epithelium (Valenta, 2005). To improve the residence time of delivery systems in the vagina, mucoadhesive formulations could be developed using polymers as alginate, hyaluronic acid and derivatives, pectin,
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chitosan. . .which could interact with mucus by different mechanisms (Peppas and Buri, 1985; Gu et al., 1988; Woodley, 2001). Mucoadhesion phenomena are not well understood (Edsman and Hägerström, 2005; Smart, 2005) and several theories have been proposed to explain mucoadhesion (Peppas and Sahlin, 1996; Ahuja et al., 1997; Edsman and Hägerström, 2005; Khutoryanskiy, 2010) such as electronic, adsorption, wetting, fracture, mechanical and diffusion theories. Drug absorption and mucoadhesion efficiency are dependent on the physicochemical properties of the polymers and the drugs used in the formulation (Gurny et al., 1984; Duchene et al., 1988; Alambar and Akhrul, 2005; Asane et al., 2008). The use of probiotics, defined as ‘‘live microorganisms which when administered in adequate amounts confer a health benefit on the host’’ (Reid and Bruce, 2003; FAO/WHO, 2001) offers a potential alternative approach to health restoration and maintenance in the vaginal tract (Reid, 2001). Another way to operate consists in using prebiotics in order to provide nutrients which stimulate the Lactobacilli growth to the detriment of pathogens. The use of prebiotics is a more recently introduced concept. Many studies have shown prebiotics benefits on human health not only on gastrointestinal microflora but also anticancer and immune effects (Macfarlane et al., 2006; Lenoir-Wijnkoop et al., 2007). Rousseau et al. (2005) studied prebiotic effects on the urogenital ecosystem. They evaluated fructo- and gluco-oligosaccharides series varying by their osidic linkages. The results showed that oligosaccharides are good candidates to prevent vaginal infections. To our knowledge, nowadays no pharmaceutical studies were described for the encapsulation of probiotics associated with prebiotics for vaginal use while probiotics encapsulation is a process widely used in food applications in order to protect microorganisms against stomach acidity (Champagne and Fustier, 2007). One of the most common method used for probiotic encapsulation is based on the gelation of alginate in contact with calcium ions (Krasaekoopt et al., 2003). The main objective of this study was to encapsulate probiotic strains and prebiotic into bioadhesive pectinate-hyaluronic acid microparticles dedicated to the formulation of a vaginal delivery system. In this study, the encapsulation system could enhance the effects of Lactobacillus sp. and protect them during drying process and storage. As alginate, low methyl pectin (LM pectin) forms a gel in contact with divalent cations such as Ca2+ ions. It is generally accepted that the gelation mechanism of LM pectin relies mainly on the well-known ‘‘egg-box model’’ (Grant et al., 1973). However, some authors (Racape et al., 1989) criticized this model for gelation of amidated pectins. The gel formation of LM pectin depends on several factors: pH, nature and concentration of the cross-linking agent (Ca2+, Zn2+. . .), concentration and composition of LM pectin, presence or not of sugar (Löfgren et al., 2006; Bourgeois et al., 2006; Assifaoui et al., 2010). We chose to associate calcium-pectinate with hyaluronic acid sodium salt (HA) in order to combine their bioadhesive properties suitable for vaginal use (Valenta, 2005; Asane et al., 2008; Rinaudo, 2008). Moreover, the two polymers can repair vaginal dryness. Pectin can also decrease vaginal residue (Caswell and Kane, 2002) and the combination of HA with Döderlein’s bacillus could improve Lactobacillus sp. effectiveness to normalize vaginal ecosystem (Baldacci, 1997; Valenta, 2005). The first step of this study was to prepare unloaded bioadhesive microparticles based on pectinate/HA mixture with a size between 100 and 200 lm. These preliminary experiments allowed to welldefine the experimental factors influencing the microparticle size distribution as well as their experimental range. Then, an experimental design has been realized to improve the operating conditions of the microencapsulation process and obtain bioadhesive microparticles able to encapsulate the pro- and prebiotics.
2. Materials and methods 2.1. Materials Calcium chloride (CaCl2), hyaluronic acid sodium salt (HA), porcine stomach mucins, potassium hydroxide (KOH), calcium hydroxide (Ca(OH)2), bovine serum albumin, lactic acid, acetic acid, glycerol, urea, glucose, EDTA, TweenÒ 80, SpanÒ 80, methylene blue and Eriochrome black T were purchased from Sigma–Aldrich (St. Quentin Fallavier, France). Low methyl pectin was obtained from CargillÒ (St. Germain en Laye, France). Vegetal oil (C8/C10) and stearate sucroesters were obtained from Stéarinerie Dubois (Boulogne-Billancourt, France). Fructo-oligosaccharides (prebiotics), FOS ActilightÒ 950S and 950P were a gift of Béghin Meiji (Marckolsheim, France). Four selected commercial probiotic strains including Lactobacillus rhamnosus, L. salivarius, L. brevis and L. plantarum were a gift of NebraskaÒ cultures (Walnut Creek, California, USA). The probiotic organisms were maintained individually at 20 °C. The Man Rogosa Sharpe (MRS) broth was purchased from BD DifcoTM (Pont de Claix, France). Acridine orange was purchased from Alfa Aesar GmbH & Co KG (Karlsruhe, Germany). 2.2. Microencapsulation process The microencapsulation process was performed according to an emulsification/gelation method (Capela et al., 2007; Sheu et al., 1993). Briefly, 37.5 ml of an aqueous solution made of sterile LM pectin, hyaluronic acid sodium salt (HA) and fructooligosaccharides (FOS) was prepared in presence or not of probiotics. Different polymer and FOS concentrations were studied: from 2% to 7% (w/v) for LM pectin, from 0.5% to 1% (w/v) for HA and from 2% to 10% (w/ v) for FOS. This aqueous solution was then emulsified into a vegetal oil containing surfactant (Polysorbate 80 or stearate sucroester) for 30 min. Different impeller types at 1200 rpm were evaluated as stirring device as well as an ultra-turraxÒ at 13,500 rpm. Then, 150 ml of calcium chloride (0.1 M or 0.2 M) were added under stirring (with an addition rate ranging from 10 to 20 ml/min) inducing the formation of calcium pectin/HA microparticles. The addition of an aqueous solution containing 0.9% (w/v) of NaCl allowed the emulsion breakage. Then, the oil layer was drained, the microparticles were removed from the aqueous phase by filtration and washed twice with saline water. Microparticles were stored at +4 °C into physiological water. 2.3. Microparticle characterization 2.3.1. Size The size distribution of pectin/HA/Ca2+ microparticles were measured by laser light scattering (Mastersizer 2000, Malvern Instruments, UK). 2.3.2. Microparticle morphology Microparticles were observed by optical microscopy after adding methylene blue inside the loaded microparticle suspension. A blue coloration of probiotic loaded-microparticles was observed while unloaded microparticles remained uncolored. This technique was also very helpful to quickly verify probiotic encapsulation. 2.4. Complexometric determination of Ca2+ ions amount into pectin/ HA beads Ca2+ ions entrapped into microparticles were quantified by ethylene diaminetetraacetic acid (EDTA) titration. EDTA is a chelating agent complexing with most metal ions. The equivalence point is
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detected through the use of the indicator Eriochrome Black T which is itself a chelating agent. After microparticle formulation and oil removal, the aqueous phase was isolated. 10 ml of the aqueous phase and 10 ml basic equimolar buffer with a pH = 9.50 (ammonium hydroxide, chloride hydroxide) were introduced into Erlenmeyer flask. Before titration some droplets of an ethanol solution of Eriochrome Black T were added and the aqueous phase was titrated by EDTA (0.1 M). 2.5. Bioadhesion evaluation by rheological measurements Rheological measurements were conducted on pectinate or pectinate/HA gels in order to evaluate their bioadhesive properties. Pectin or pectin/HA solutions were prepared by dispersing the required amounts of polymers in 0.9% (w/v) NaCl. All samples were stirred at +80 °C for 1 h. About 5% (w/v) of FOS were added to each sample. The pH of all solutions was set to 4.2. Then, pre-heated solution of CaCl2 was added in the polymer solutions under stirring during 1 min. The gels obtained were stored at room temperature during 48 h before rheological measurements. Rheological measurements were carried out at +37 °C using a Bohlin VOR controlled rate rheometer using a parallel plate (PP30) measuring system. After the identification of the linear viscoelastic region at 1 Hz, gels were investigated over a frequency range from 0.03 to 5 Hz. All samples were prepared in duplicate and each of them was analyzed five times. 2.6. Probiotic characterization 2.6.1. Evaluation of probiotic bacteria encapsulation using an acridine orange fluorescence labeling This technique consisted in labeling probiotics with acridine orange, which is a nucleic acid selective fluorescent cationic dye useful for cell cycle determination. Acridine orange which is excited under blue light, has the faculty to be cell-permeable and to interact with DNA by intercalation and RNA by electrostatic interactions. DNA intercalated acridine orange fluoresces at yellow-green wavelength (525 nm) while RNA electrostatically bounded acridine orange fluoresces at red ones (>630 nm). Before the encapsulation process, an acridine orange solution was added to the probiotic suspension under magnetic stirring during 10 min. Then probiotics were isolated by filtration, washed twice and introduced in the polymer solution. Once microparticles were formed, the suspension was observed by confocal microscopy. Acridine orange fluorescence technique is an easy and sensitive method for screening biological samples. However in some case it could be difficult to well-distinguish between yellow-green and red colors. Moreover the contact time and the concentration of acridine orange could spark off these color variations (Maki and Remsen, 1981) and the colors observed not always translate the bacteria activity (Quinn, 1984). Therefore this technique has been completed by the pour plate technique using MRS agar so as to confirm or not the viability of probiotic strains. 2.6.2. Enumeration of probiotic bacteria To determine the viable counts of the encapsulated probiotic bacteria, microparticles were dispersed in phosphate buffer. After complete release of probiotics, serial dilutions were prepared with peptone water and culture was plated by the pour plate technique using MRS agar. Plates were incubated in jars Anaerobac anaerobiosis-generating systems at +37 °C for 48 h. Platting was carried out in triplicate. 2.6.3. Kinetic release of probiotics from microparticles The release profile of probiotics from microparticles of the improved formulation was followed during 24 h. The in vitro release
medium was a simulated vaginal fluid (SVF) at pH 4.2 whose composition is described in Table 1 (Owen and Katz, 1999; Das Neves et al., 2008). About 0.100 g of microparticles were dispersed under magnetic stirring at +37 °C in 9 ml of SVF since daily vaginal secretions were estimated in a range of 1–11 ml (Owen and Katz, 1999) After incubation time of 30 min, 1, 3, 5, 7, 16 and 24 h, all samples were filtered in order to only recuperate a bacterial suspension. The enumeration of the released probiotic bacteria was then performed by the pour plate technique using MRS agar as described previously. Plates were incubated in Anaerobac jars (anaerobiosis-generating systems) at +37 °C for 48 h. Platting was carried out in duplicate. 3. Results and discussion 3.1. Preliminary experiments The influence of several formulation and process parameters (Table 2) was first evaluated on the size distribution of unloaded microparticles. 3.1.1. Process parameters From a technical point of view, the choice of an impeller type depends on the direction (radial or axial) of the flow to be generated. The encapsulation process was made of two very different steps: (i) an emulsion step for which shear forces (radial dispersion) are needed in order to produce relatively monodisperse and small droplets and (ii) a gelation step requiring axial flow to mix efficiently the added Ca2+ solution with pectin gel. Therefore, three impeller types were evaluated to prepare the microparticles: a four-blade impeller inducing axial dispersion, a Rushton turbine and an ultra-turraxÒ homogenizer suitable to generate high shear forces. Similar size distributions (with a mean size around 200 lm) were measured for microparticles prepared by using a four blade impeller or a Rushton turbine (results not shown). As expected, the use of an ultra-turraxÒ homogenizer at 13,500 rpm during the emulsification step allowed to obtain smaller microparticles with a mean size comprised between 20 and 60 lm. This size range made the removal of microparticles from the oil phase much more difficult with only a small portion of them being recovered. Moreover homogenization using ultra-turraxÒ led to local heating of the emulsion that could alter probiotic viability. From an energetical point of view, the four-blade impeller was the best stirring system able to produce satisfying microparticles and to preserve probiotics during the encapsulation process. 3.1.2. Oil type and polymer:oil ratio The oil type was found to play an important role to avoid aggregate formation during the gelation step. Different oil types have been studied: mineral oils (paraffin, cyclopentasiloxan, pentan) and vegetal oils (sunflower, triglyceride mixtures). Oil viscosity
Table 1 Simulated vaginal fluid composition. Component quantity (g/l) Porcine gastric mucin (type II) NaCl KOH Ca(OH)2 Bovine serum albumin Lactic acid Acetic acid Glycerol Urea Glucose
15 3.51 1.40 0.222 0.018 2.00 1.00 0.16 0.4 5.0
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Table 4 HLB of surfactant used.
Formulation and process parameters HLB
Oil type (mineral and vegetal oil) Polymer:oil ratio [Polymers] (HA and LM pectin) Impeller type [Prebiotics] Type of surfactant [Surfactant]
reported in Table 3 had a significant effect on the size distribution and the morphology of the microparticles. Low viscosity oils (0– 10 mPa s) led to very large microparticles with a mean diameter of around 4 mm and more. For oil viscosity higher than 50 mPa s, as for sunflower and paraffin oils, the mean diameter of the microparticles ranged from 200 up to 700 lm. In this case, the formation of aggregates was observed, microparticles filtration became uneasy and the oil excess was difficult to remove. The triglyceride (TG) mixture allowed a good compromise between the formulated microparticle size and their ease of use for the entire formulation process. Indeed regular microparticles were obtained without any aggregate formation during the calcium addition. Elimination of the oil phase was easier and only a few oily residues remained in the final microparticle suspension. Moreover, no significant difference on the particle mean diameter was observed by varying the C18/C10 composition of the triglyceride mixture (70/20; 65/35; 55/45). A polymer:oil ratio of 1:4 was selected so as to obtain a good dispersion of the polymer solution inside the oil phase and to facilitate the CaCl2 solution addition. For larger polymer amounts (corresponding to polymer:oil ratios of 1:2 and more), the emulsification of the polymer solution became more difficult as well as the addition of calcium solution leading to the formation of microparticle aggregates. 3.1.3. Concentration and type of surfactants Once the oil phase defined, polymer in triglycerides emulsions have been formulated by using different surfactants in order to estimate the required HLB of the triglyceride mixture. Two surfactant couples were studied: TweenÒ 80/SpanÒ 80 and two sucroesters (5S and 15S) whose HLB are reported in Table 4. For each surfactant couple, the studied HLB range was the following: 5, 5.5, 6, 6.5, 7, 8, 9, 10 and 15. In each case, W/O emulsions were obtained. Coalescence between droplets was observed by optical microscopy for emulsions stabilized by TweenÒ 80/SpanÒ 80 couple while sucroesters led to stable emulsions. The required HLB of the TG mixture was 5 corresponding to the sucroester 5S HLB. In the classical emulsification/gelation process (Sheu et al., 1993), TweenÒ 80 surfactant is used to facilitate the removal of the oil phase at the end of the process for an easier microparticle separation. We decided to replace this hydrophilic surfactant by the sucroester 5S to preferentially stabilize the W/O emulsion. The microparticles exhibited monomodal size distribution which were quite similar with a mean diameter about 110 lm for TweenÒ
Table 3 Viscosity of each oil type measured at +25 °C. Oil type
Viscosity (mPa s)
Pentan Cyclopentasiloxan Triglyceride mix Sunflower Paraffin
0.225 4 28–3158 58 170
TweenÒ 80
Span 80
Sucroester 5S
Sucroester 15S
15
4.3
5
15
80 formulation and 99 lm for sucroester 5S ones (results not shown). The type of surfactant did not significantly influence the particle size distribution but had an impact on microparticle shape. The use of sucroester 5S inside the oil phase led to the formation of stable emulsions and spherical and regular microparticles while microparticle aggregates were observed with TweenÒ 80. We assumed that the emulsion stabilized with 5S sucroester did not immediately break with the CaCl2 solution addition as in the case of TweenÒ 80 emulsion. 3.1.4. Polymer concentration The bioadhesivity of different pectin gels prepared with or without HA was evaluated by a rheological method based on the comparison of their storage moduli. These measurements were generally realized in presence or not of mucins so as to estimate the interactions between polymer gels and mucin. The interaction parameter DG0 is calculated from the equation:
G0mix ¼ G0p þ G0m þ DG0 G0mix ,
G0p
ð1Þ
G0m
where and are the elastic (storage) moduli of the mixture, the polymer and the mucins, respectively. The absolute synergism parameter DG0 is the elastic component which translates the interaction between polymers and mucin (Rossi et al., 1995; Hägerström et al., 2000; Andrews and Jones, 2006). The elastic modulus of the mucin dispersion being negligible, the following simplified equation is generally used to calculate DG0 :
G0mix ¼ G0p þ DG0
ð2Þ
Moreover, in the case of the calcium-pectinate gels, mucins could interact with Ca2+ ions reducing the interaction between pectin and Ca2+ ions that led to weakly gels. Indeed, divalent cations could be bound by the negative charges of mucus sialic acid (Park and Robinson, 1985). In particular, Hägerström et al. (2000) have shown that mucin addition could lead to the destruction of gel structure. Therefore we decided not to use mucins in our study assuming that an increase of the storage modulus (i.e. DG0 > 0) translates an increase of the adhesion effect. Different concentrations of polymers were used to prepare calcium-pectinate gels: LM pectin concentration ranged from 2% up to 7% (w/v) and HA from 0.5% to 1% (w/v). As expected, the obtained results for the pectin/Ca2+ gels (Fig. 1) showed an increase of the storage modulus G0 with the pectin concentration. The size distributions of microparticles prepared with 3%, 5% or 7% (w/v) pectin are presented in Fig. 2. A monomodal distribution was observed for 3% (w/v) of pectin with a mean diameter of 102 lm. Higher pectin concentrations led to more viscous solutions decreasing the stirring efficiency and inducing multimodal size distributions with higher mean diameters of 128 and 303 lm for 5% and 7% (w/v) pectin, respectively. Then, rheological measurements have been performed on different gels of pectin/Ca2+ and pectin/Ca2+/HA in order to evaluate the influence of HA addition on adhesive properties of pectin gels. HA was added at 0.5% or 1% (w/v). The evolution of G0 modulus presented in Fig. 1 shows that HA addition inside pectin/Ca2+ gels could enhance the gel adhesive properties. Higher adhesion effect was observed for pectin/Ca2+/0.5% (w/v) HA gels whatever pectin concentration. For 1% (w/v) of HA, the conclusions were not so clear. An improvement of the adhesion effect was clearly observed for gel with at least 5% (w/v) of pectin. For 4% pectin, no significant
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Fig. 1. Evaluation of the adhesive properties of (j) LM pectin/Ca2+ gels, (4) LM pectin/Ca2+/HA 0.5% (w/v) gels and (s) LM pectin/Ca2+/HA 1% (w/v) gels by measuring the evolution of G’ as a function of pectin concentration.
Fig. 2. Influence of pectin concentration on microparticle size distribution: (dash line) pectin 3% (w/v), (dotted line) pectin 5% (w/v) and (dotted dash line) pectin 7% (w/v).
evolution of G0 was observed and for lower pectin concentrations, the addition of HA at 1% (w/v) induced a decrease of G0 indicating the formation of weaker gels. Calcium diffusion in an alginate solution leads to gelation by complexing with the polymer carboxylic groups which is translated by a significant loss of water against the current of Ca2+ flux, during the gelation process (Yotsuyanagi et al., 1987). A study on alginate–hyaluronate gel and beads (Oerther et al., 1999) investigated the gel formation between alginate, HA and Ca2+ ions showing that HA addition reduced the dehydration effect of gels and that interactions between HA and Ca2+ ions could be dependent on the composition of the polymer mixtures. HA addition could disturb the diffusion of Ca2+ ions and also the equilibrium of water and Ca2+ ions content in the gel. In this way HA addition leads to a modification of gel structure and reduced hardness of gel. Furthermore Ca2+ ions diffusion in alginate gel (without HA) appeared slower than in gels containing a little amount of HA. They recommended to use a high molecular weight and a moderate concentration of HA so as to make gel easier. To our knowledge, no study evaluated interaction between HA and pectin, but as alginate presents the same properties as pectin with Ca2+ ions, similar phenomena inside pectin/Ca2+/HA gels could be suggested. In our study, the gels obtained with 0.5% (w/v) HA presented stronger structure than with 1% (w/v) HA according to the G0 values (Fig. 1). We could conclude that addition of 0.5% (w/v) HA had no
real impact on pectin gelation process. At 1% (w/v) HA and especially for low pectin concentration, we could expect that Ca2+ diffusion was significantly limited leading to important hydration effect and a weaker gel structure. The gels could get stronger structure with an increase of pectin concentration until counterbalancing the HA effect for pectin concentrations above 4% (w/v). These observations confirm the results of Oerther et al. (1999). To obtain efficient pectin gelation and adhesion effect at low pectin concentration, HA concentration was set at 0.5% (w/v) for the following experiments, especially for the experimental design. The addition of HA in pectin gels used for the preparation of microparticles did not significantly influence the particle size distribution (Fig. 3). However, larger particles have been obtained with HA addition (102 lm for 0% (w/v) HA, 156 lm for 0.5% (w/ v) HA, 136 lm for 1% (w/v) HA) probably due to the increase of the dispersed phase viscosity.
3.1.5. Prebiotics concentrations Prebiotics are known as nutrients which stimulate the Lactobacilli growth to the detriment of pathogens (Lee and Salminen, 2009). Prebiotics were also added inside microparticles in order to complete the probiotic effect. The influence of prebiotics concentration in pectin gels on microparticle size distribution was investigated.
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Fig. 3. Influence of HA concentration on microparticle size distribution: (dotted line) pectin 3% (w/v), (dotted dash line) pectin 3% (w/v)/HA 0.5% (w/v) and (dash line) pectin 3% (w/v)/HA 1% (w/v).
A
B
Fig. 4. Prebiotic influence on microparticle size distribution with : (A) pectin 3% (w/v)/HA 1% (w/v) and FOS : (continuous line) 0%, (dotted line) 2% (w/v), (dash line) 5% (w/v), (dotted dash line) 10% (w/v) ; (B) pectin 3% (w/v) with 5% (w/v) FOS and (continuous line) HA 1% (w/v) or (dash line) HA 0.5% (w/v).
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3.2. Experimental design
Table 5 Determination of Ca2+ ions amount inside microparticles. Microparticles composition
Ca2+ mole ion number (for 1 g of LM pectin) (mmol)
3% (w/v) of Pectin [CaCl2] = 0.1 M 3% (w/v) of Pectin + 5% (w/v) of FOS [CaCl2] = 0.1 M 3% (w/v) of Pectin + 5% (w/v) of FOS + 0.5% HA [CaCl2] = 0.1 M 3% (w/v) of Pectin + 5% (w/v) of FOS + 0.5% HA [CaCl2] = 0.2 M
3.7 ± 0.1 3.8 ± 0.1 2.9 ± 0.1 5.4 ± 0.1
Table 6 Set parameters for experimental design. Set parameter Oil type Polymer:oil ratio [HA] [Prebiotics] Impeller type Surfactant
Triglyceride mix (C8/C10) 1:4 0.5% (w/v) 5% (w/v) Four blade impeller Sucroesters 5S (HLB = 5)
The size distribution of microparticles made of 3% (w/v) pectin and 1% (w/v) HA in presence or not of prebiotics were first measured (Fig. 4A). Prebiotic concentration had a significant impact on the particle size distribution with a mean diameter of 261 lm for 2% (w/v) prebiotics, 177 lm for 5% (w/v) prebiotics and 199 lm for 10% (w/v) prebiotics. The addition of prebiotics increased the microparticle size especially for 2% and 10% prebiotics. According to our previous results, prebiotics could have an impact on HA faculty to reduce gel dehydration promoting microparticle swelling. We did not find any explanations describing the real impact of prebiotics on the microparticle size distribution. Fu and Rao (2001) have studied sucrose effect on low methyl pectin gelation. They observed that sucrose had the faculty to reduce the water content promoting polymer–polymer interactions rather than polymer–solvent interactions. Furthermore, sucrose could stabilize the cross-linking junction in gel thanks to additional hydroxyl groups that promoted hydrogen bonds to immobilize free water. HA concentration being set at 0.5% (w/v) for mucoadhesion reasons, we decided to compare the size distribution of microparticles obtained with 5% (w/v) prebiotics and 1% or 0.5% (w/v) of HA. No significant difference on size distribution was observed (Fig. 4B). Therefore this prebiotic concentration was chosen for further experiments. 3.1.6. Complexometric determination of Ca2+ ions into microparticles The quantification of Ca2+ involved in the pectin/HA mixture gelation was performed for some formulations. The results obtained are reported in Table 5 and showed that prebiotic addition did not significantly affect the LM pectin gelation. On the contrary, HA addition led to a decrease of Ca2+ ions inside the microparticles suggesting, as discuss above, that HA presence could slow down Ca2+ ions diffusion and therefore the gelation process. As expected, the use of a more concentrated CaCl2 solution, allowed the penetration inside the microparticles of a larger content of calcium ions.
The preliminary experiments allowed the setting of several parameters which are reported in Table 6: the type of oil and surfactant, the oil/polymer ratio, HA and prebiotics concentrations and the impeller type. We also well-defined the experimental range of the remaining operating parameters (Table 7) whose effect on microparticle properties (size and encapsulation efficiency) was investigated by the means of an experimental design. For all these experiments, probiotics strains were added to the polymer solution contrary to the preliminary experiments. Considering the great number of experimental factors (7 parameters), a two-levels fractional factorial design was used for screening purpose allowing the identification of 16 runs (Table 8). This experimental design was built by aliasing the main effects of the factors studied with high-order interactions assumed to be negligible. The chosen factors with their coded levels 1, 0 and + 1 are summarized in Table 7. The measured responses were the microparticle mean diameter d [0.5] and the probiotic encapsulation rate (Table 8). The use of screening design consists in approximating the responses to be measured (Y) by a first-order model whose coefficients bi correspond to the main effects of the experimental factors (Xi) to be studied (Box et al.,2005):
Y ¼ b0 þ
7 X
bi X i
ð3Þ
i¼1
The coefficients bi were calculated by least squares linear regression (Fig. 5) and the results were analyzed by NEMRODWÒ software version 2000 (Nemrodw, LPRAI, Marseille, France). The constant b0 corresponds to the average of the responses for the 16 runs. The factors strongly influencing the response are those having the highest absolute value for bi. The sign of the coefficient shows how the factor influences the response: if the coefficient is negative, the response decreases when the factor ranges from the coded level (1) to (+1); the contrary is obtained if the coefficient is positive. Then, coefficient signs allow the determination of the best coded level for each factor. The coefficient calculation (Fig. 5) highlighted two significant factors influencing the microparticle mean size which varied from 87 to 729 lm depending on the operating conditions applied: the stirring rate (X1) and the pectin concentration (X3). The stirring rate (X1) had to be increased at the higher level (1200 rpm) so that the particle mean size decreased below 380 lm. An increase in pectin quantity (X3) led to an increase in particle mean size. This effect could be attributed to a reduction of stirring efficiency induced by the increase of the organic phase viscosity. Moreover, bimodal distributions were obtained for microparticles made with 5% (w/ v) of pectin while monomodal distributions characterized the formulations based on 3% (w/v) of pectin (Fig. 6). The pectin concentration at 3% (w/v) was then preferred. By considering the standard deviation about 15 lm of the D (0.5) estimated through the replication of experiments, results surprisingly showed no significant effect of the surfactant concentration (X5) on the particle size for the experimental range studied.
Table 7 Design factors and their levels.
Levels 1 0 +1
Stirring rate (rpm)
Emulsification time (min)
Pectin (%)
CaCl2 (M)
Surfactant (%)
Gelation time (min)
Addition rate of CaCl2 (ml/min)
X1 720 960 1200
X2 10 20 30
X3 3 4 5
X4 0.1 0.15 0.2
X5 0.02 0.11 0.2
X6 10 15 20
X7 10 15 20
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Table 8 Experimental design. No. exp.
Stirring rate (rpm)
Emul. time (min)
[Pectin] (%(w/v))
[CaCl2] (M)
[Surfactant] (%(w/v))
Gel. time (min)
Addition rate of CaCl2 (ml/min)
D (0.5) (lm)
Encapsulation rate (108 CFU/g microparticles)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
720 1200 720 1200 720 1200 720 1200 720 1200 720 1200 720 1200 720 1200
10 10 30 30 10 10 30 30 10 10 30 30 10 10 30 30
3 3 3 3 5 5 5 5 3 3 3 3 5 5 5 5
0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2
0.02 0.2 0.2 0.02 0.02 0.2 0.2 0.02 0.2 0.02 0.02 0.2 0.2 0.02 0.02 0.2
10 20 20 10 20 10 10 20 10 20 20 10 20 10 10 20
10 20 10 20 20 10 20 10 20 10 20 10 10 20 10 20
229 87 132 137 640 357 729 195 453 171 377 104 686 379 539 239
1.53 1.43 1.38 1.53 1.42 1.56 1.44 1.58 1.74 2.41 1.37 1.59 1.51 1.69 1.96 1.72
Fig. 5. The main effects of the experimental factors on the particle mean diameter.
Fig. 7. Methylene blue labeled microparticles obtained with pectin 3% (w/v), HA 0.5% (w/v) and FOS 5% (w/v) (experiment no. 4) observed by optical microscopy (40). Fig. 6. Pectin concentration influence on the size distribution of probiotic-loaded microparticles obtained from experiment no. 4 (dotted line) with pectin 3% (w/v) or from experiment no. 14 (dotted dash line) with pectin 5% (w/v).
The other factors had reduced influence compared to the stirring rate and the pectin concentration. The measured values for the second response, the probiotic encapsulation rate, varied between 1.4 108 and 2.4 108 CFU/g
of microparticles. These results were considered quite similar according to the experimental error estimated by the replication of experiments with a standard deviation about 0.6 108 CFU/g of microparticles. This indicates that the operating factors studied did not affect significantly the probiotic encapsulation rate in the experimental range studied.
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Fig. 8. Acridine orange labeled microparticles obtained with pectin 3% (w/v), HA 0.5% (w/v) and FOS 5% (w/v) (experiment no. 4) observed by (A) transmission and (B) confocal microscopy (20).
Fig. 9. Percentage of probiotics released in vitro from microparticles obtained with pectin 3% (w/v), HA 0.5% (w/v) and FOS 5% (w/v) (experiment no. 4) as a function of time.
The operating conditions for experiment no. 4 were consistent with the conclusions drawn from the experimental design study and led to interesting properties for our application being a good compromise between the mean diameter (137 lm) and the size distribution of the microparticles. The reproducibility of this formulation was then verified. Fig. 7 represents a microscopic observation of the improved formulation containing methylene blue showing an effective encapsulation of the probiotics. Indeed for probiotic-loaded microparticles, methylene blue remained fixed on probiotics that led to a blue coloration of microparticles. For unloaded microparticles no coloration was observed. Moreover, acridine orange fluorescence was used to specifically label Lactobacillus sp. (as described in Section 2). This labeling allowed confirming that probiotic strains were inside the microparticles and not in the external phase of the suspension (Fig. 8). Finally, the release profile of probiotics from microparticles of the improved formulation (experiment no. 4) was evaluated. Fig. 9 shows a sustained release of the probiotics during 10 h.
The probiotics were totally released after 16 h incubation and a stabilization of the bacteria population was observed between 16 and 24 h. The percentage values obtained exceeding 100 could reflect the beginning of the probiotic proliferation. This type of release profile is interesting for a vaginal application since microparticles allowed a sustained release of probiotics followed by a bacteria proliferation. This aspect will be more investigated from the final form (vaginal tablets incorporating-microparticles) in a future work. 4. Conclusion Pro-and pre-biotics-loaded bioadhesive microparticles made of HA and low methyl pectin have successfully been developed for a pharmaceutical use. The operating conditions allowing the preparation of favorable microparticles for probiotic encapsulation have been determined through an experimental design. The microparticle obtained exhibited adhesive properties as shown by the rheological measurements and a sustained release of probiotics. In
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