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aCenter for Advanced Biomaterials for H

Tecnologia, Via Largo Barsanti e Matteu

raffaele.vecchione@iit.itbInterdisciplinary Research Center of Bioma

Tecchio, 80 80125, Naples, ItalycNanophysics - Istituto Italiano di Tecnolog

Italy

† Electronic supplementary informationFig. S1–S3, and Tables S1–S6. See DOI: 10

Cite this: Nanoscale, 2014, 6, 9300

Received 27th April 2014Accepted 27th May 2014

DOI: 10.1039/c4nr02273d

www.rsc.org/nanoscale

9300 | Nanoscale, 2014, 6, 9300–9307

Tunable stability of monodisperse secondary O/Wnano-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

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

a few weeks to several months. The above range of concentrations has been investigated using a

fluorescence-based approach with new insights into the coating evolution.

Introduction

The development of nano-carriers capable of conveying andreleasing bioactive substances is currently creating greatinterest and it is becoming one of the most important objectivesof, above all, food, cosmetics and pharmaceutical industries.1–5

For instance, the capability to encapsulate materials on thenanoscale can impact the food industry in facing the enormouschallenge of making food both healthy and tasty,6,7 orenhancing biodistribution of nutraceuticals;3 the cosmeticsindustry, by improving the stability of various cosmetic ingre-dients (like unsaturated fatty acids and vitamins), enhancingpenetration of certain ingredients (such as vitamins),8

increasing the efficacy and tolerance of UV lters on the skinsurface and making the product more appealing.9 Especially inthe drug delivery eld, the ideal size of drug carriers is in thenanometer range, particularly below 200 nm.10 Moreover,monodispersity is another fundamental requirement to providestability over time and to avoid any erratic behavior.11 Forinstance, in pharmaceutical formulations the maximum

ealth Care@CRIB, Istituto Italiano di

cci 53, 80125, Naples, Italy. E-mail:

terials, CRIB, University Federico II, P.le

ia (IIT), Via Morego 30, 16163, Genova,

(ESI) available: Experimental section,.1039/c4nr02273d

volume percentage of carriers in the micrometer range has tomeet the specications of the European and United States ofPharmacopeia. Oil-in-water (O/W) nano-emulsions, consistingof small lipid droplets dispersed in an aqueous medium, can besuitable 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 todissolve large quantities of hydrophobics and protect theircargo from hydrolysis and enzymatic degradation makes nano-emulsions 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 andthey present destabilization mechanisms such as creaming,sedimentation, coalescence, occulation and Ostwaldripening.22 Caruso and his group proposed a preparationstrategy that permits the formation of monodisperse emulsionsby lling with oil some polyelectrolyte nano-capsules previouslyprepared via layer by layer (LBL) around a solid template.23

Apart from the advantage of gaining control over emulsionmonodispersity, this method is very time-consuming, not suit-able for large-scale production and limited in the scaling downof the emulsion size. Another strategy to improve the emulsionstability – proposed by McClements and his group – is toproduce secondary emulsions obtained with a polyelectrolytethin layer, adsorbed through the interaction with an ionicemulsier of opposite charge standing on the surface of oildroplets.24 Such amethod, even simpler than previous one, doesnot completely solve the stability issue. The same author alsoproposed to use a high-pressure homogenizer to process the

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emulsion together with a food grade polymer to stabilize O/Wnano-emulsions, but the nal colloidal systems presented awide size distribution, predictably leading to fast destabiliza-tion.25 It was then attempted to improve emulsion stability bycross-linking the polymer coating,26 but the emulsion sizeshowed a signicant increase over time due to destabilization.Therefore, emulsion monodispersion and stability still remainan open issue in the eld of colloidal systems. In this study weimplemented a new post-processing method based on multi-re-dispersion at high pressure that allowed us to improvesecondary emulsion homogeneity and polymer coatingcompactness and – therefore – monodispersion and stability.We assumed that the pressure is able to densify the poly-electrolyte coating27 and that the high pressure homogenizer isable to disrupt aggregates and gain in monodispersion. Weproved this method on two different classes of biodegradablepolyelectrolytes, such as chitosan as an example of poly-saccharides and polylysine as an example of polypeptides. Inparticular, in the case of the emulsion coated with chitosan, itsbehavior was monitored in terms of stability over time for morethan one year. We were able to obtain unprecedented, ultra-stable emulsions at high polymer concentrations. The absenceof aggregations at these high concentrations was due to themultiple re-dispersion process originally implemented in thiswork. By varying the polymer concentration, it was possible tocontrol the level of polymer coating and obtain nano-emulsionswith tunable stability: the less the polymer, the less the stability.In the case of stabilization with polymer multi-layers, it is reallycrucial to “understand” the level of polymer coating around theemulsion to avoid excess in solution. The Z-potential method iscommonly used,24 but it does not provide a precise evaluation ofthe coating conditions. Herein, we also tried to solve this issueby studying the evolution of the polymer deposition aroundprimary emulsions through uorescence emission tests thatprovided novel results regarding the saturation regime.

Fig. 1 Emulsion size at different processing cycles and surfactantamounts.

Results and discussionNano-emulsion preparation

One condition affecting the emulsion stability is the degree ofpolydispersion at production time. Therefore, the rst effortwas to identify the right conditions to reach not only the desiredsizes (down to 100 nm), but also the right PDI (<0.1). We chose abiocompatible formulation consisting of soy-bean oil andLipoid E80 lecithin. Then, we aimed to cover a dimensionalrange below 200 nm and down to 100 nm and used differentlecithin concentrations. In particular, O/W primary emulsionsat ve different lecithin concentrations were prepared accord-ing to the procedure reported in the Experimental section. Theuse of a high-pressure homogenizer allowed reaching aminimum size for each amount of surfactant. Specically, wecombined 3 single passes through the homogenizing chamberwith further continuous recycling steps. Indeed, by using singlecycles, only the rst three were really effective in size reduction.Aer these three single passes we combined continuous steps.DLS analysis made it possible to observe how the variation in

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the number of single passes, continuous steps and lecithinconcentrations affected the droplet size (Fig. 1).

Aer a signicant decrease in the emulsion size with thesingle cycles, a further decrease during continuous processingup to a plateau was observed in all the formulations. A repro-ducible size was attained at each formulation, provided that thesame processing chamber was used (an example is reported inthe ESI section, Table S1†), from around 160 nm, with thelowest concentration of lecithin (L1), to around 80 nm with thehighest one (L5). The latter was slightly smaller than the L4-based emulsion, meaning that a further increase in the amountof lecithin would have been ineffective. Each system was verystable while measuring, as demonstrated by the very low stan-dard deviation. As for the size, PDI decreased with the numberof cycles, but increased with the amount of surfactant, eventhough it remained below 0.1. The precise values of the size,PDI, and Z-potential for all the samples are summarized in theESI section (Table S2†). These results show the capability tocover a dimensional range from 200 to less than 100 nm withpolydispersion indices below 0.1. These are quite unique andinteresting dimensional features for bio-applications, asmentioned in the Introduction.10 However, low molecularweight emulsiers like lecithin, fast in coating and stabilizingthe emulsion while processing, are typically not able to protectemulsions for long time.28 We performed stability tests over therst two and half months on these ve emulsions to verify thedegree of destabilization. The results are summarized in the ESIsection (Table S3†). In particular, at room temperature thesmallest emulsions (L4 and L5) were quite stable, while the rstthree changed a lot maybe due to the higher deformability,which, in turn, can induce fast coalescence. In addition, asshown in the table, we tested emulsions kept at 4 �C. In thiscase, also the three largest emulsions (L1, L2, L3) gainedstability, maybe due to the slowing down of the droplets, whichreduces the efficacy of hits and the following coalescence. Tosignicantly improve emulsion stability, as anticipated in theIntroduction, we resorted to homogeneous and densied poly-mer coatings. It is well known that emulsions with a surface

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charge can be stabilized using a polyelectrolyte coating; they arethen called secondary emulsions, and their stabilization capa-bility depends on the concentration of the polymer itself.24 Ifconcentration is not appropriate, stability can be even lower. Inthis work, not only we varied the polymer concentration, butalso attempted to identify an optimal process procedure toimprove stability. The main issue in this kind of protection isthat, while mixing polyelectrolyte solution with emulsion, thepolymer starts to coat the emulsion before reaching a uniformdistribution, thus producing bridging occulation. Sometimes,to prevent coating while mixing, it is proposed to play with thepH of the system so that – aer mixing – the pH is brought to avalue where the two components have an opposite charge tointeract and allow polymer coating in a more uniform way.29

Such a procedure is quite limiting since it can be applied only topolymers that can change their charge according to the pH andall the other components have to remain unaffected by the widerange of pH explored. Herein, aer the preparation of secondaryemulsions, they were re-dispersed at high pressure (around 700bar) according to the procedure reported in the Experimentalsection. This way, we expected a re-dispersion of the polymerand a densication of the coating with predictable improve-ment in monodispersion and stability. In particular, we per-formed and compared single and double re-dispersionprocesses one month apart to evaluate the long-term stability ofthe emulsion in terms of the size and PDI constancy over time.

Monodispersion and stability analysis

As an example, the evolution of the size over time for L1 (large),L2 (medium) and L4 (small) based double re-processedsecondary emulsions is shown in Fig. 2 (all the other data are inthe ESI section, in Table S4†).

It is worth noticing that the size of the double re-processedsamples is slightly larger (a few nm) than the starting primaryemulsions, meaning no aggregations compared with single

Fig. 2 Dimensional behavior over time for L1, L2, and L4 basedsecondary emulsions re-dispersed twice at different chitosanconcentrations.

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re-processed and even more with non-re-processed samples.The trend of the results and their reproducibility for all the veformulations demonstrate their consistency. As a note, datarelated to double re-processed samples start aer 1 month,which corresponds to their birth. For what concerns polymerconcentration, it is clear that long-term stability increases withit. Fig. 2 shows this stability trend with the polymer concen-tration regardless of the starting primary emulsion size. Inparticular, polymer concentrations needed to give stabilitywere slightly dependent on the emulsion size, as it waspredictable considering the different surface areas; the smallerthe droplets, the larger the surface area, and therefore thepolymer amount needed. Specically, a low concentration ofchitosan brought to a faster destabilization than that of theprimary emulsions; the lower the concentration, the faster thede-mixing, as expected. At polymer concentrations below 0.01wt%, secondary emulsions re-processed one time were readilyde-mixed, and therefore not suitable for further re-dispersiontreatments. On the other hand, emulsion stability improvedthe increasing polymer concentration due to an increasingcoverage of the emulsions, as discussed later. It has to beremarked that high polymer concentrations are typicallyexcluded due to aggregations24 that we also noticed in differentdegrees in single and – even more – in no re-dispersionprocesses (see Fig. S2 in the ESI section†). Conversely, wenoticed no aggregation in double re-dispersion systems, due toan enhanced homogenization of the polymer in solution. Itwas thus possible to obtain unprecedented ultrastablesecondary emulsions at high polymer concentrations. Mono-dispersion and short-term stability were also noticed for lowerconcentrations of polymers due to re-dispersion on one sideand high pressure on the other. In synthesis, the double re-processed samples behaved better than the single re-processedones and much better than the non-processed ones, both interms of the size and possible destabilization time (the same isfor PDI, see Table S4†). For the non-re-processed samples, thedestabilization trend was already evident in the rst month;therefore, no further data were collected aer this time. Due tothe high monodispersion feature and the immiscibility of thetwo liquids, the Ostwald mechanism26 was also avoided, thusallowing for a prolonged stability.

To prove the versatility of the proposed method, we alsotested a polypeptide – polylysine (PLL) – as a positive poly-electrolyte in place of chitosan. As shown in Table S5,† it ispossible to lower the PDI below 0.1 and obtain the minimumsize of the secondary emulsion once the polymer concentrationhas been xed (e.g., PLL/oil ¼ 0.02 wt/wt), only aer the doublere-dispersion process. We expect also in this case animprovement in long term stability for multi-re-dispersedsecondary emulsions, having gained in terms of mono-dispersion and having densied the polymer coating. More-over, in the case of a lower polymer concentration (PLL/oil ¼0.0125 wt/wt), therefore minimizing any excess of polymer insolution, it was possible to effectively deposit a polyelectrolytelayer of heparin thanks in particular to the monodispersionand stability features gained with the multi-re-dispersionprocess (see always Table S5†).

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To support the DLS analysis, we analyzed double re-pro-cessed L1, L2 and L4 secondary emulsions coated with chitosan(0.1 wt%) by uorescence microscopy. This analysis was per-formed using chitosan labeled with FITC (see ESI section† forlabelling procedure). It was necessary to carry out a STEDanalysis because of the expected small size, as demonstrated bythe comparison with a confocal analysis (Fig. 3).

The results are consistent with the analysis carried out in theconventional way by DLS. Even though this analysis was per-formed aer a few weeks, the monodispersion feature stayedunchanged.

Fig. 4 Emission analysis by using a spectrofluorimeter on secondaryemulsions at different FITC-labeled chitosan concentrations, toidentify the saturation concentration (top); emission comparison withfree FITC-labeled chitosan (down).

Saturation method by spectrouorimetry

To clarify the reason behind the tunable stability of thesecondary emulsions at different polymer concentrations, weexplored the saturation of the polymer around the emulsion. Inthe literature, the Z-potential method is usually proposed toevaluate this condition,30 but it has proved inaccurate. Here, inorder to estimate the amount of polymer coating aroundprimary emulsions, we measured the uorescence emission ofemulsions (e.g., on L4 template), coated with chitosan-FITC atincreasing polymer concentrations (Fig. 4).

Fig. 3 Fluorescence microscopy analysis by confocal and STED on L1,L2, and L4 based secondary emulsions. (a) and (b) are the respectiveconfocal and STED images of L4 based secondary emulsions; theboxed areas are enlarged in the insets in (c) (top confocal and bottomSTED), the white arrows show where the line profile in (c) has beenmeasured. (d) and (e) are the respective confocal and STED images ofL2 based secondary emulsions; the boxed areas are enlarged in theinsets in (f) (top confocal and bottom STED), the white arrows showwhere the line profile in (f) has been measured. (g) and (h) are therespective confocal and STED images of L4 based secondary emul-sions; the boxed areas are enlarged in the insets in (i) (top confocal andbottom STED), and the white arrows show where the line profile in (i)has beenmeasured. In all the line profiles (c), (f), and (i) the green line isa multi peak gaussian fit of the intensity green points measured on theconfocal image, while the red line is a multi peak Lorentian fit of theintensity red points measured on the STED image.

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Fluorescence was normalized with respect to the polymerconcentration itself. An initial increase of the emission wasobserved up to a maximum (at around 0.0175 wt%) due to thevariation of the charge surrounding FITC followed by adecreasing curve, which instead is characteristic of the freepolymer in solution. By making a comparison between thesesamples and free FITC-labeled chitosan at the same concen-trations, the decreasing behavior is evident in both cases.However, the slope of the two curves is different; only atconcentrations above 0.1 wt% the two curves overlap showingan identical behavior. We attributed the difference in the slopeto an incomplete coverage of the primary emulsion, whichwould explain the progressive improvement of the stabilitypreviously described from 0.01 to 0.1 wt%. The maximumvalue of the curve can be associated with a strong saturation ofthe oil surface combined with no signicant polymer in solu-tion, allowing effective deposition of a further polymer – as weproved experimentally using heparin. Indeed, higher concen-trations of chitosan were not suitable for further polymerdeposition, meaning a signicant excess of polymer in solu-tion. Therefore, this analysis shows the evolution of polymerdeposition on primary emulsions. More precisely, we assessedthat saturation cannot be associated with one concentration,but it happens in a wide range of concentrations due to theequilibrium between the polymer in solution and the polymeron the emulsion. Moreover, the identication of the switchingis much more accurate when compared to the Z-potentialmethod.30

Applicability features

Another analysis on our secondary emulsions regarded theevaluation of the micrometric emulsion percentage. In the case

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Table 1 Oil volume% between 5 and 50 mm in size in the case of L4 fora primary emulsion in the first 2 weeks and for secondary double re-dispersed emulsions at the three highest concentrations of polymersafter around 7.5 months

SampleDays aerpreparation

Oil volume %between 5 and 50 mm

Template 0 0.0012Template aer 14 days 14 0.062Monolayer 0.1 wt% CT 230 0.056Monolayer 0.175 wt% CT 230 0.032Monolayer 0.25 wt% CT 230 0.049

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of a low amount of micrometric particles, they are not visibleat the DLS and a more accurate analysis is necessary. Thelower is the amount of micrometric emulsion percentage, thehigher is the shelf life of the product. As a comparison, inFig. 5 we show the number of micrometric particles for aprimary emulsion of small size (�80 nm) during the rst twoweeks aer preparation and the same emulsion stabilizedby double re-dispersion at the last three polymerconcentrations.

The destabilization of the emulsion template is evident inthe micrometer range when compared to double re-dispersedsecondary emulsions; to give a number, we took into accountthe maximum value specied by USP (Chapter 729) in the eldof lipid injectable emulsions for parenteral nutrition, which is avery stringent condition. The specication imposes that thevolume percentage of particles between 5 and 50 mm stays below0.05%. By making this quantication, we found that – aer 2weeks – emulsions with no polymer coating had already over-come the specied threshold, while the stabilized secondaryemulsions still met the specication standard aer around 7.5months (see Table 1) at polymer concentrations above 0.1 wt%.This is an enormous advantage from the application point ofview, since these emulsions present a very high shelf life, as alsodemonstrated experimentally.

In addition, in vitro tests of chitosan based secondaryemulsions on broblast cells showed no signicant cytotoxicityeffect, as reported in more detail in the ESI section (Fig. S3†).Other in vitro tests on cancer cells show an evident cytotoxiceffect on the cells due to the selective action of the curcumincargo used in these experiments.

Taking advantage of the spectrouorimeter analysis and bythe monodispersion and short-term stability features of theabove described secondary emulsions, a polymer layer ofheparin (an example of natural polyelectrolyte of oppositecharge) was effectively deposited on the chitosan coated emul-sion (see ESI, Table S6†). This is a proof that our secondaryemulsions, obtained with polylysine (Table S5†) or with chito-san (Table S6†), can be used as a template for layer by layer,which is one of the most interesting methods to build uppolymer nanocapsules for drug delivery.31

Fig. 5 Particle size distribution by granulometric analysis.

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Experimental

Primary oil in water nano-emulsions were prepared by using ahigh-pressure homogenizer (Microuidics M110PS) and aformulation based on soy-bean oil (density at 20 �C of 0.922 gml�1) and Lipoid E80 lecithin (egg lecithin powder 80–85%enriched with phosphatidyl choline (PC) and 7–9.5% contentin phosphatidyl ethanolamine (PE) as per the manufacturer'sspecications); both products, were purchased from Lipoidand used without further purication. Both soy-bean oil andLipoid E80 are pharmagrade products and used in parenteralnutrition because of their well known biodegradability. Milli-pore® Milli-Q water was used for the preparation of all emul-sions and solutions. The emulsions were then coated withchitosan (CT, LMW 90–150 kDa, DDA 84% determined via 1H-NMR), a linear random copolymer of D-glucosamine and N-acetyl-D-glucosamine (pKa � 6.7), which was puried before use(see the ESI section†) and with poly-L-lysine hydrobromide(mol wt 4000–15 000 by viscosity), both purchased from Sigma-Aldrich. Some secondary emulsions were further coated withheparin (heparin sodium salt from porcine intestinal mucosa,17–19 kDa) also purchased from Sigma Aldrich. Both chitosanand heparin are natural polysaccharides largely used inbiomedical applications,32,33 while polylysine is a naturalpolypeptide again already studied for the preparation of drugdelivery nanocarriers.34

Sizes were measured by dynamic light scattering (DLS), aswell as by uorescence microscopy both confocal and STED(stimulated emission depletion). Zeta potential measurementswere also carried out to verify charge switching aer polymerdeposition. For the uorescence analysis, chitosan was chemi-cally labeled with uorescein 5(6)-isothiocyanate (FITC, Mw ¼389.38 g mol�1) (details are available in the ESI section†) andpurchased from Sigma Aldrich. 1,4-Diazabicyclo[2.2.2]octane(dabco) (Mw ¼ 112.17 g mol�1) antifade, also purchased fromSigma Aldrich, was used to avoid the bleaching phenomenon ofFITC. FD3510 tissue culture dishes with cover glass bottomwereused to allow STED observation of the samples. Some emulsionswere also characterized in terms of the size by loading themwith piperine (97%, Mw ¼ 285.34) as an example of nutraceuticand Nile Red (Mw ¼ 318.37 g mol�1) as an example of thelipophilic model drug and in terms of cytotoxicity with anotherbioactive nutraceutic like curcumin (from Curcuma longa

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(turmeric), powder, Mw ¼ 368.38). All these lipophiliccompounds were purchased from Sigma-Aldrich and usedwithout further purication.

Primary emulsion

First, the oil phase was prepared by adding a certain amount ofLipoid E 80, previously weighed on a high-precision scale, to amass of soy-bean oil. The oil phase was mixed (for 10 min at atemperature of 60 �C and at a speed of 500 rpm) using a highspeed blender (RZR 2102 control, Heidolph), and then, aerreaching room temperature, it was sonicated for 1.5 min (asonication amplitude of 60%; both pulse-on and pulse-off of 10s) with an immersion sonicator (Ultrasonic Processor VCX500Sonic and Materials). These two steps were repeated until leci-thin was completely dissolved. To obtain the pre-emulsion, theoil phase was added dropwise to a weighted amount of aqueousphase (Milli-Q water) and mixed using the immersion sonicatorduring the rst 3 min (a sonication amplitude of 70%; a pulse-on and a pulse-off respectively of 10 and 5 s) and then foranother 5 min under the same conditions. In order to avoid pre-emulsion overheating (leading to thermal agitation of pre-emulsion droplets, thus increasing the probability of coales-cence during sonication), preparation was made in a largebeaker in which an ice jacket was created around a smallerbeaker containing the oil-phase. The pre-emulsions were nallypassed at 2000 bar through the high-pressure valve homoge-nizer (Microuidics M110PS) for the rst three individual cyclesto strongly reduce the initial size and then continuously by re-lling the reservoir for several steps, thus recycling the productwithout any loss of volume and taking out small volumes ofemulsion at 50, 100, 150 and 200 steps for DLS. This methodwas used for the preparation of oil-in-water emulsions at 20wt% of oil phase in water. Each emulsion with different oil andlecithin concentrations was classied according to the LipoidE80/oil phase ratio, as reported in Table 1.

Secondary emulsion

Primary emulsions were coated with chitosan, which is posi-tively charged and therefore susceptible to be electro-staticallydeposited on oil droplets stabilized with lecithin, which isnegative in charge. To evaluate the optimal concentration ofchitosan to be deposited, solutions at several concentrations ofchitosan (0.0025, 0.005, 0.00875, 0.0125, 0.025, 0.05, 0.0875,0.125, 0.250, 0.5, 0.875, and 1.25 wt%), were prepared in 0.1 Macetic acid Milli-Q water. 9 ml of these solutions were diluted1 : 4 with Milli-Q water to 36 ml and brought to pH 4 with asmall addition of NaOH 6 M solution. Then, 9 ml of the emul-sion diluted to 5 wt% of oil phase were added quickly undervigorous stirring and kept under stirring for 15 min to allowuniform chitosan deposition. The emulsion was diluted 1 : 5with chitosan solution (thus the nal oil concentration was 1wt%, whereas nal chitosan concentrations were 1/5 of theinitial). The emulsions with the rst layer of chitosan werepassed through a high-pressure valve homogenizer at 700 barfor 100 continuous steps, and re-processed aer one monthunder the same conditions. Each system was analyzed over time

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and stored at room temperature. In order to demonstrate theversatility of the method, primary emulsions were also coatedwith polylysine, but at a neutral pH, and some results at onepolymer concentration (0.02 wt%) are reported.

Z-potential measurements

The Z-potential of emulsions and emulsion-based nano-capsules was determined using a particle electrophoresisinstrument (Zetasizer zs nano series ZEN 3600, MalvernInstruments Ltd., Malvern, U.K.). The Z-potential is determinedby measuring the direction and velocity of droplet movementsin a well-dened electric eld. Prior to analysis, emulsions werediluted up to a droplet concentration of approximately 0.025wt% using Milli-Q water; secondary emulsions were diluted upto the same concentration using in the case of chitosan anacetic acid Milli-Q water solution (pH 4, 20 mM). For doublecoated nano-emulsions the same dilutions in MilliQ water wasused. The Z-potential analysis was carried out by setting 50 runsfor each measurement.

Particle size measurements

The particle size distribution was measured using a laserdynamic light scattering instrument (l ¼ 632.8 nm). Thisinstrument correlates the uctuations of intensity I(t) of scat-tered light during the time with the Brownian motion ofcolloidal particles and then with the diffusion coefficient Drelated to the hydrodynamic radius Rh of the particles. Adetecting angle of 173� was used. All the samples were dilutedup to a droplet concentration of approximately 0.025 wt%, byusing Milli-Q water in the case of primary emulsions and anacetic acid Milli-Q water solution (pH 4, 20 mM) in the case ofchitosan based secondary emulsions. For double coated nano-emulsions, the same dilutions in MilliQ water were used. Adefault refractive index ratio (1.52) and 5 runs for eachmeasurement (1 run lasting 100 s) were used in the calculationsof the particle size distribution.

Spectrouorimetric analysis

In order to evaluate polymer coverage on primary emulsions, auorescence spectroscopy analysis was performed (EnSpireMultimode Plate Reader 2300-0000, Perkin Elmer). 100 ml ofsample at different FITC-labeled chitosan concentrations (0.01,0.00175, 0.0025, 0.005, 0.01, 0.0175, 0.025, 0.05, 0.1, 0.175, and0.25 wt%) were loaded in a multi-well (3 replicates for eachsample were prepared) and irradiated with a light with awavelength of 488 nm (the light coming from a ash lamp isltered in order to obtain the desired wavelength). The uo-rescence signal was detected at 510 nm. The used number ofashes was 100.

STED analysis description

A procedure was also developed to allow observation of all thesamples with the STED high resolution using a uorescenceoptical microscope. Each sample was diluted 1 : 50 to a nal oilconcentration of 0.02 wt% in a Eppendorf with a 20 mM acetic

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acid buffer solution at pH 4 and was put in a FD3510 dish for 30min to allow it to adhere to the surface of the dish. Aer that, 3washes with a 5 wt% dabco antifade solution were performedand at the end the central part of the dish was le full of dabcoduring sample observation. As previously mentioned, dabcoantifade was needed to avoid the bleaching effect of the FITC-labeled chitosan. Samples were imaged with a Leica TCS STED-CW microscope (Leica-Microsystems, Mannheim, Germany).The resolution of the microscope was estimated to be <80 nm.For each different sample, we acquired 10 images with a eld ofview of 25.6 � 25.6 mm for a pixel size of 25 � 25 nm. The STED-CW beam power was 430 mW, measured at the objective backfocal plane. The analysis of the images was carried out usingImagej soware.35

Conclusions

We demonstrated that it is possible to produce secondaryemulsions showing unparalleled long-term stability with acontrol in size depending on the formulation (down to 100 nm),and PDI (#0.1), thanks to the implementation of a novel multire-dispersion process at high-pressure. In particular, we showedthat, in order to get very stable secondary emulsions, a specicconcentration is not sufficient, as it must be combined with anoptimized post-process. Such a process was identied as amulti-re-dispersion over time by homogenizing at high pressure(around 700 bar) in order to densify the polymer coating andgain in monodispersion and – therefore – in further stability.Multi-re-processed secondary emulsions with no aggregationsat a much higher level of coating than what is typical (above 0.1wt%) were proved to satisfy USP specications even aer severalmonths, unlike bare emulsions showing a high shelf life. Byvarying the polymer concentration, we demonstrated capabilityto tune emulsion stability over time – from a few weeks toseveral months – thanks to an increase in the coating level. Weprovided further evidence of this concept since we investigatedthe saturation regime by employing a uorescence approach,with new insights into the evolution of the polymer depositionaround primary emulsions. Starting from this understandingand taking advantage of the monodispersion and short-termstability features (at low polymer concentrations) of oursecondary emulsions, we were also able to easily identify theproper polymer concentration and effectively deposit a secondpolymer layer, opening up new possibilities for the build-up ofbiodegradable nanocapsules via layer by layer.

Acknowledgements

The authors thank Valentina La Tilla for the graphical abstractrealization and Chiara Cosenza for Matlab representation ofsome results. Moreover, the authors thank Vincenzo Quagliar-iello for the in vitro tests, Vincenzo Calcagno for chitosanlabeling with FITC, and Annalisa Marsiglia for reproducibilityspectrouorimeter tests. Finally, the authors thank Sig. Ric-cardo Cossi from QiTech for the micrometric analysis evalua-tion by using Accusizer and Mrs Roberta Infranca for herprecious and careful proofreading.

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