Nanoscale View Article Online
PAPER
Published on 29 May 2014. Downloaded by Université Laval on 30/11/2014 01:59:09.
Cite this: Nanoscale, 2014, 6, 9300
View Journal | View Issue
Tunable stability of monodisperse secondary O/W nano-emulsions† R. Vecchione,*ab U. Ciotola,b A. Sagliano,b P. Bianchini,c A. Diasproc and P. A. Nettiab Stable and biodegradable oil in water (O/W) nano-emulsions can have a huge impact on a wide range of bio-applications, from food to cosmetics and pharmaceuticals. Emulsions, however, are immiscible systems unstable over time; polymer coatings are known to be helpful, but an effective procedure to stabilize monodisperse and biodegradable O/W nano-emulsions is yet to be designed. Here, we coat biodegradable O/W nano-emulsions with a molecular layer of biodegradable polyelectrolytes such as polysaccharides – like chitosan – and polypeptides – like polylysine – and effectively re-disperse and densify the polymer coating at high pressure, thus obtaining monodisperse and stable systems. In particular, focusing on chitosan, our tests show that it is possible to obtain unprecedented ultra-stable
Received 27th April 2014 Accepted 27th May 2014
O/W secondary nano-emulsions (diameter sizes tunable from 80 to 160 nm and polydispersion indices below 0.1) by combining this process with high concentrations of polymers. Depending on the polymer concentration, it is possible to control the level of coating that results in a tunable stability ranging from
DOI: 10.1039/c4nr02273d
a few weeks to several months. The above range of concentrations has been investigated using a
www.rsc.org/nanoscale
fluorescence-based approach with new insights into the coating evolution.
Introduction The development of nano-carriers capable of conveying and releasing bioactive substances is currently creating great interest and it is becoming one of the most important objectives of, above all, food, cosmetics and pharmaceutical industries.1–5 For instance, the capability to encapsulate materials on the nanoscale can impact the food industry in facing the enormous challenge of making food both healthy and tasty,6,7 or enhancing biodistribution of nutraceuticals;3 the cosmetics industry, by improving the stability of various cosmetic ingredients (like unsaturated fatty acids and vitamins), enhancing penetration of certain ingredients (such as vitamins),8 increasing the efficacy and tolerance of UV lters on the skin surface and making the product more appealing.9 Especially in the drug delivery eld, the ideal size of drug carriers is in the nanometer range, particularly below 200 nm.10 Moreover, monodispersity is another fundamental requirement to provide stability over time and to avoid any erratic behavior.11 For instance, in pharmaceutical formulations the maximum
a
Center for Advanced Biomaterials for Health Care@CRIB, Istituto Italiano di Tecnologia, Via Largo Barsanti e Matteucci 53, 80125, Naples, Italy. E-mail: raff[email protected]
b
Interdisciplinary Research Center of Biomaterials, CRIB, University Federico II, P.le Tecchio, 80 80125, Naples, Italy
c Nanophysics - Istituto Italiano di Tecnologia (IIT), Via Morego 30, 16163, Genova, Italy
† Electronic supplementary information (ESI) available: Experimental section, Fig. S1–S3, and Tables S1–S6. See DOI: 10.1039/c4nr02273d
9300 | Nanoscale, 2014, 6, 9300–9307
volume percentage of carriers in the micrometer range has to meet the specications of the European and United States of Pharmacopeia. Oil-in-water (O/W) nano-emulsions, consisting of small lipid droplets dispersed in an aqueous medium, can be suitable as the basis of many kinds of foods (e.g., milk, cream, beverages, dressings, etc.),12,13 as well as cosmetics (lotions, transparent milks, and crystal-clear gels).14–18 Their ability to dissolve large quantities of hydrophobics and protect their cargo from hydrolysis and enzymatic degradation makes nanoemulsions ideal vehicles for the purpose of drug delivery.19–21 However, even though stabilized by the use of emulsiers, emulsions do not typically have a sufficiently long shelf life and they present destabilization mechanisms such as creaming, sedimentation, coalescence, occulation and Ostwald ripening.22 Caruso and his group proposed a preparation strategy that permits the formation of monodisperse emulsions by lling with oil some polyelectrolyte nano-capsules previously prepared via layer by layer (LBL) around a solid template.23 Apart from the advantage of gaining control over emulsion monodispersity, this method is very time-consuming, not suitable for large-scale production and limited in the scaling down of the emulsion size. Another strategy to improve the emulsion stability – proposed by McClements and his group – is to produce secondary emulsions obtained with a polyelectrolyte thin layer, adsorbed through the interaction with an ionic emulsier of opposite charge standing on the surface of oil droplets.24 Such a method, even simpler than previous one, does not completely solve the stability issue. The same author also proposed to use a high-pressure homogenizer to process the
This journal is © The Royal Society of Chemistry 2014
View Article Online
Published on 29 May 2014. Downloaded by Université Laval on 30/11/2014 01:59:09.
Paper
emulsion together with a food grade polymer to stabilize O/W nano-emulsions, but the nal colloidal systems presented a wide size distribution, predictably leading to fast destabilization.25 It was then attempted to improve emulsion stability by cross-linking the polymer coating,26 but the emulsion size showed a signicant increase over time due to destabilization. Therefore, emulsion monodispersion and stability still remain an open issue in the eld of colloidal systems. In this study we implemented a new post-processing method based on multi-redispersion at high pressure that allowed us to improve secondary emulsion homogeneity and polymer coating compactness and – therefore – monodispersion and stability. We assumed that the pressure is able to densify the polyelectrolyte coating27 and that the high pressure homogenizer is able to disrupt aggregates and gain in monodispersion. We proved this method on two different classes of biodegradable polyelectrolytes, such as chitosan as an example of polysaccharides and polylysine as an example of polypeptides. In particular, in the case of the emulsion coated with chitosan, its behavior was monitored in terms of stability over time for more than one year. We were able to obtain unprecedented, ultrastable emulsions at high polymer concentrations. The absence of aggregations at these high concentrations was due to the multiple re-dispersion process originally implemented in this work. By varying the polymer concentration, it was possible to control the level of polymer coating and obtain nano-emulsions with tunable stability: the less the polymer, the less the stability. In the case of stabilization with polymer multi-layers, it is really crucial to “understand” the level of polymer coating around the emulsion to avoid excess in solution. The Z-potential method is commonly used,24 but it does not provide a precise evaluation of the coating conditions. Herein, we also tried to solve this issue by studying the evolution of the polymer deposition around primary emulsions through uorescence emission tests that provided novel results regarding the saturation regime.
Results and discussion
Nanoscale
the number of single passes, continuous steps and lecithin concentrations affected the droplet size (Fig. 1). Aer a signicant decrease in the emulsion size with the single cycles, a further decrease during continuous processing up to a plateau was observed in all the formulations. A reproducible size was attained at each formulation, provided that the same processing chamber was used (an example is reported in the ESI section, Table S1†), from around 160 nm, with the lowest concentration of lecithin (L1), to around 80 nm with the highest one (L5). The latter was slightly smaller than the L4based emulsion, meaning that a further increase in the amount of lecithin would have been ineffective. Each system was very stable while measuring, as demonstrated by the very low standard deviation. As for the size, PDI decreased with the number of cycles, but increased with the amount of surfactant, even though it remained below 0.1. The precise values of the size, PDI, and Z-potential for all the samples are summarized in the ESI section (Table S2†). These results show the capability to cover a dimensional range from 200 to less than 100 nm with polydispersion indices below 0.1. These are quite unique and interesting dimensional features for bio-applications, as mentioned in the Introduction.10 However, low molecular weight emulsiers like lecithin, fast in coating and stabilizing the emulsion while processing, are typically not able to protect emulsions for long time.28 We performed stability tests over the rst two and half months on these ve emulsions to verify the degree of destabilization. The results are summarized in the ESI section (Table S3†). In particular, at room temperature the smallest emulsions (L4 and L5) were quite stable, while the rst three changed a lot maybe due to the higher deformability, which, in turn, can induce fast coalescence. In addition, as shown in the table, we tested emulsions kept at 4 C. In this case, also the three largest emulsions (L1, L2, L3) gained stability, maybe due to the slowing down of the droplets, which reduces the efficacy of hits and the following coalescence. To signicantly improve emulsion stability, as anticipated in the Introduction, we resorted to homogeneous and densied polymer coatings. It is well known that emulsions with a surface
Nano-emulsion preparation One condition affecting the emulsion stability is the degree of polydispersion at production time. Therefore, the rst effort was to identify the right conditions to reach not only the desired sizes (down to 100 nm), but also the right PDI (
PAPER
Published on 29 May 2014. Downloaded by Université Laval on 30/11/2014 01:59:09.
Cite this: Nanoscale, 2014, 6, 9300
View Journal | View Issue
Tunable stability of monodisperse secondary O/W nano-emulsions† R. Vecchione,*ab U. Ciotola,b A. Sagliano,b P. Bianchini,c A. Diasproc and P. A. Nettiab Stable and biodegradable oil in water (O/W) nano-emulsions can have a huge impact on a wide range of bio-applications, from food to cosmetics and pharmaceuticals. Emulsions, however, are immiscible systems unstable over time; polymer coatings are known to be helpful, but an effective procedure to stabilize monodisperse and biodegradable O/W nano-emulsions is yet to be designed. Here, we coat biodegradable O/W nano-emulsions with a molecular layer of biodegradable polyelectrolytes such as polysaccharides – like chitosan – and polypeptides – like polylysine – and effectively re-disperse and densify the polymer coating at high pressure, thus obtaining monodisperse and stable systems. In particular, focusing on chitosan, our tests show that it is possible to obtain unprecedented ultra-stable
Received 27th April 2014 Accepted 27th May 2014
O/W secondary nano-emulsions (diameter sizes tunable from 80 to 160 nm and polydispersion indices below 0.1) by combining this process with high concentrations of polymers. Depending on the polymer concentration, it is possible to control the level of coating that results in a tunable stability ranging from
DOI: 10.1039/c4nr02273d
a few weeks to several months. The above range of concentrations has been investigated using a
www.rsc.org/nanoscale
fluorescence-based approach with new insights into the coating evolution.
Introduction The development of nano-carriers capable of conveying and releasing bioactive substances is currently creating great interest and it is becoming one of the most important objectives of, above all, food, cosmetics and pharmaceutical industries.1–5 For instance, the capability to encapsulate materials on the nanoscale can impact the food industry in facing the enormous challenge of making food both healthy and tasty,6,7 or enhancing biodistribution of nutraceuticals;3 the cosmetics industry, by improving the stability of various cosmetic ingredients (like unsaturated fatty acids and vitamins), enhancing penetration of certain ingredients (such as vitamins),8 increasing the efficacy and tolerance of UV lters on the skin surface and making the product more appealing.9 Especially in the drug delivery eld, the ideal size of drug carriers is in the nanometer range, particularly below 200 nm.10 Moreover, monodispersity is another fundamental requirement to provide stability over time and to avoid any erratic behavior.11 For instance, in pharmaceutical formulations the maximum
a
Center for Advanced Biomaterials for Health Care@CRIB, Istituto Italiano di Tecnologia, Via Largo Barsanti e Matteucci 53, 80125, Naples, Italy. E-mail: raff[email protected]
b
Interdisciplinary Research Center of Biomaterials, CRIB, University Federico II, P.le Tecchio, 80 80125, Naples, Italy
c Nanophysics - Istituto Italiano di Tecnologia (IIT), Via Morego 30, 16163, Genova, Italy
† Electronic supplementary information (ESI) available: Experimental section, Fig. S1–S3, and Tables S1–S6. See DOI: 10.1039/c4nr02273d
9300 | Nanoscale, 2014, 6, 9300–9307
volume percentage of carriers in the micrometer range has to meet the specications of the European and United States of Pharmacopeia. Oil-in-water (O/W) nano-emulsions, consisting of small lipid droplets dispersed in an aqueous medium, can be suitable as the basis of many kinds of foods (e.g., milk, cream, beverages, dressings, etc.),12,13 as well as cosmetics (lotions, transparent milks, and crystal-clear gels).14–18 Their ability to dissolve large quantities of hydrophobics and protect their cargo from hydrolysis and enzymatic degradation makes nanoemulsions ideal vehicles for the purpose of drug delivery.19–21 However, even though stabilized by the use of emulsiers, emulsions do not typically have a sufficiently long shelf life and they present destabilization mechanisms such as creaming, sedimentation, coalescence, occulation and Ostwald ripening.22 Caruso and his group proposed a preparation strategy that permits the formation of monodisperse emulsions by lling with oil some polyelectrolyte nano-capsules previously prepared via layer by layer (LBL) around a solid template.23 Apart from the advantage of gaining control over emulsion monodispersity, this method is very time-consuming, not suitable for large-scale production and limited in the scaling down of the emulsion size. Another strategy to improve the emulsion stability – proposed by McClements and his group – is to produce secondary emulsions obtained with a polyelectrolyte thin layer, adsorbed through the interaction with an ionic emulsier of opposite charge standing on the surface of oil droplets.24 Such a method, even simpler than previous one, does not completely solve the stability issue. The same author also proposed to use a high-pressure homogenizer to process the
This journal is © The Royal Society of Chemistry 2014
View Article Online
Published on 29 May 2014. Downloaded by Université Laval on 30/11/2014 01:59:09.
Paper
emulsion together with a food grade polymer to stabilize O/W nano-emulsions, but the nal colloidal systems presented a wide size distribution, predictably leading to fast destabilization.25 It was then attempted to improve emulsion stability by cross-linking the polymer coating,26 but the emulsion size showed a signicant increase over time due to destabilization. Therefore, emulsion monodispersion and stability still remain an open issue in the eld of colloidal systems. In this study we implemented a new post-processing method based on multi-redispersion at high pressure that allowed us to improve secondary emulsion homogeneity and polymer coating compactness and – therefore – monodispersion and stability. We assumed that the pressure is able to densify the polyelectrolyte coating27 and that the high pressure homogenizer is able to disrupt aggregates and gain in monodispersion. We proved this method on two different classes of biodegradable polyelectrolytes, such as chitosan as an example of polysaccharides and polylysine as an example of polypeptides. In particular, in the case of the emulsion coated with chitosan, its behavior was monitored in terms of stability over time for more than one year. We were able to obtain unprecedented, ultrastable emulsions at high polymer concentrations. The absence of aggregations at these high concentrations was due to the multiple re-dispersion process originally implemented in this work. By varying the polymer concentration, it was possible to control the level of polymer coating and obtain nano-emulsions with tunable stability: the less the polymer, the less the stability. In the case of stabilization with polymer multi-layers, it is really crucial to “understand” the level of polymer coating around the emulsion to avoid excess in solution. The Z-potential method is commonly used,24 but it does not provide a precise evaluation of the coating conditions. Herein, we also tried to solve this issue by studying the evolution of the polymer deposition around primary emulsions through uorescence emission tests that provided novel results regarding the saturation regime.
Results and discussion
Nanoscale
the number of single passes, continuous steps and lecithin concentrations affected the droplet size (Fig. 1). Aer a signicant decrease in the emulsion size with the single cycles, a further decrease during continuous processing up to a plateau was observed in all the formulations. A reproducible size was attained at each formulation, provided that the same processing chamber was used (an example is reported in the ESI section, Table S1†), from around 160 nm, with the lowest concentration of lecithin (L1), to around 80 nm with the highest one (L5). The latter was slightly smaller than the L4based emulsion, meaning that a further increase in the amount of lecithin would have been ineffective. Each system was very stable while measuring, as demonstrated by the very low standard deviation. As for the size, PDI decreased with the number of cycles, but increased with the amount of surfactant, even though it remained below 0.1. The precise values of the size, PDI, and Z-potential for all the samples are summarized in the ESI section (Table S2†). These results show the capability to cover a dimensional range from 200 to less than 100 nm with polydispersion indices below 0.1. These are quite unique and interesting dimensional features for bio-applications, as mentioned in the Introduction.10 However, low molecular weight emulsiers like lecithin, fast in coating and stabilizing the emulsion while processing, are typically not able to protect emulsions for long time.28 We performed stability tests over the rst two and half months on these ve emulsions to verify the degree of destabilization. The results are summarized in the ESI section (Table S3†). In particular, at room temperature the smallest emulsions (L4 and L5) were quite stable, while the rst three changed a lot maybe due to the higher deformability, which, in turn, can induce fast coalescence. In addition, as shown in the table, we tested emulsions kept at 4 C. In this case, also the three largest emulsions (L1, L2, L3) gained stability, maybe due to the slowing down of the droplets, which reduces the efficacy of hits and the following coalescence. To signicantly improve emulsion stability, as anticipated in the Introduction, we resorted to homogeneous and densied polymer coatings. It is well known that emulsions with a surface
Nano-emulsion preparation One condition affecting the emulsion stability is the degree of polydispersion at production time. Therefore, the rst effort was to identify the right conditions to reach not only the desired sizes (down to 100 nm), but also the right PDI (
