Heat Treatment of Poloxamer-Stabilized Triglyceride Nanodispersions

Article Cite This: Mol. Pharmaceutics 2018, 15, 3111−3120

Heat Treatment of Poloxamer-Stabilized Triglyceride Nanodispersions: Effects and Underlying Mechanism Katrin Göke,†,‡ Elin Roese,† and Heike Bunjes*,†,‡ †

Institut für Pharmazeutische Technologie, Technische Universität Braunschweig, Mendelssohnstraße 1, 38106 Braunschweig, Germany ‡ Zentrum für Pharmaverfahrenstechnik, Franz-Liszt-Straße 35a, 38106 Braunschweig, Germany

Mol. Pharmaceutics 2018.15:3111-3120. Downloaded from pubs.acs.org by DURHAM UNIV on 08/08/18. For personal use only.

S Supporting Information *

ABSTRACT: Lipid nanoemulsions are being investigated for the parenteral administration of poorly soluble drugs. A narrow particle size distribution in these formulations is a prerequisite for meaningful research and safe administration to patients. Autoclaving a poloxamerstabilized trimyristin nanoemulsion resulted in moderate particle growth and a strong decrease in particle size distribution width (Göke, K.; Roese, E.; Arnold, A.; Kuntsche, J.; Bunjes, H. Mol. Pharmaceutics 2016, 13, 3187.). In this work, the critical parameters for such a change upon autoclaving poloxamer 188-stabilized lipid nanodispersions were investigated to elucidate the underlying mechanism. Nanodispersions of triglycerides with esterified fatty acid chain lengths from C8 to C18 were treated at different temperatures and for varying durations. The influence of a decrease in poloxamer 188’s cloud point was tested by adding potassium chloride to the dispersions prior to autoclaving. The influence of poloxamer 188 concentration and of the type of emulsifier was investigated. The change in particle size and particle size distribution width upon heat treatment was analyzed by dynamic or static light scattering or differential scanning calorimetry. A short esterified fatty acid chain length of the triglycerides, high temperatures, and the addition of potassium chloride were key factors for particle growth up to emulsion break up, whereas the cloud point of poloxamer 188 was irrelevant. Sodium dodecyl sulfate and sucrose laurate had negative effects on emulsion stability during autoclaving. It was concluded that the increase in particle size and the decrease in particle size distribution widths upon heat treatment resulted from heat-accelerated Ostwald ripening and not from a coalescence-based process. KEYWORDS: autoclaving, particle growth, particle size distribution, Ostwald ripening, cloud point, poloxamer 188

1. INTRODUCTION

atures. Neither was the exact mechanism underlying the phenomenon elucidated in the initial study. Emulsions may only be kinetically, but never thermodynamically stable, which is why they always show a tendency to reduce their interfacial area, i.e., to increase their droplet size or even to break. Basically, particle growth in emulsions may proceed via coalescence as a result of creaming or aggregation, or by Ostwald ripening, which might be eventually followed by coalescence when the droplets have become too big.13 Coalescence is the fusion of two or more droplets to one larger particle. When two emulsion droplets come into close contact, a thin film of liquid still separates the two droplets. If the layer of emulsifier around each droplet is not able to stabilize this film, the film thins and eventually ruptures, resulting in one bigger droplet. Accordingly, coalescence is promoted by a decrease in surface tension, high kinetic energy, and low repulsive forces between particles. An increase in particle collision rate should also speed up coalescence.14

Lipid nanoparticles are under investigation as delivery systems for poorly water-soluble drugs.1 For parenteral, in particular, intravenous administration, the mean particle size in these dispersions should be below 500 nm.2 The particle size strongly influences important pharmaceutical properties of the particles like biodistribution via the bloodstream3,4 and drug loading capacity.5,6 In general, small particles with a narrow particle size distribution are desired. Various techniques exist to achieve a narrow particle size distribution, each with its own advantages and drawbacks.7−11 In a recent study, we could show that autoclaving of trimyristin nanoemulsions stabilized with poloxamer 188 (pol188; Figure 1) at 121 °C resulted in moderate particle growth and a remarkable decrease in particle size distribution width (e.g., from a z-average particle size of about 90 to 145 nm with corresponding PDI values of 0.10 and 0.04, respectively).12 Especially small particles vanished from the dispersion upon autoclaving. Heat treatment was thus considered a promising approach to achieve a narrow particle size distribution in trimyristin nanoemulsions. However, the study did not attempt to identify critical parameters for the process or assessed transferability to other lipids or temper© 2018 American Chemical Society

Received: Revised: Accepted: Published: 3111

February 23, 2018 June 4, 2018 June 15, 2018 July 10, 2018 DOI: 10.1021/acs.molpharmaceut.8b00202 Mol. Pharmaceutics 2018, 15, 3111−3120

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Molecular Pharmaceutics

The authors attributed these effects to the high cloud point of pol188 and the low interfacial tension of castor oil.19 Autoclaving cubic phase particles made from monoolein and poloxamer 407 transformed vesicular particles into larger cubic structures, which was accompanied by an increase in particle size and a decrease in particle size distribution width.21 For a very similar colloidal system, Barauskas et al.22 discovered that the particle size upon autoclaving increased almost linearly with increasing concentrations of monoolein and poloxamer 407. The addition of salt (10 mM NaCl) resulted in large aggregates upon autoclaving, which was attributed to electrostatic interactions. If the destabilizing mechanism during autoclaving was discussed in these studies, the authors suggested fusion, flocculation, or coalescence without any reference to Ostwald ripening.18−20,22 However, Ostwald ripening or molecular transfer of lipid in triglyceride dispersions is a common phenomenon and mass transfer of lipids across the continuous phase of the dispersions may even be accelerated by micelles of nonionic surfactants, which can act as carriers for the lipid.23−27 The first aim of this study was to identify critical parameters for moderate particle growth and a strong decrease in particle size distribution width for pol188-stabilized lipid nanodispersions. The second objective was to understand the underlying mechanism and, on the basis of this, to assess the transferability of the results to other lipid particle systems. In the previous study, nanoemulsions of 10% trimyristin stabilized with 5 or 12% pol188 were autoclaved at 121 °C for 15 min. In the study described in the following, we systematically varied each of these parameters, i.e., the autoclaving temperature, the duration of the heat treatment, the concentration of emulsifier and lipid, and the lipid component. On the basis of the results of these variations, we then investigated the interplay between lipid component and temperature. We also studied the effect of KCl in the continuous phase of the emulsion upon heat treatment, since the chloride anion decreases the cloud point of nonionic emulsifiers.28 The type of emulsifier was not systematically varied, since it had already been shown that many nonionic emulsifiers caused a strong increase in particle size upon autoclaving.18,19 Besides, only phospholipids, sodium oleate, pol188, polyoxyl 35 castor oil, cholesterol, and polysorbate 80 are approved emulsifiers for intravenous drug products.29 Consequently, we performed only sporadic experiments with other emulsifiers, i.e., poloxamer 407, sodium dodecyl sulfate, and sucrose laurate to address specific questions.

Figure 1. General structure of poloxamers. Poloxamer 188: a = 75− 85, b = 25−30; Poloxamer 407: a = 95−105, b = 54−60.

During emulsion degradation via Ostwald ripening, the bigger particles grow at the expense of the smaller particles. The process results from the fact that the solubility of the dispersed phase in the continuous phase increases with increasing droplet curvature, i.e., decreasing droplet size. Because of the increased solubility, the dispersed phase present in the smaller droplets dissolves in the continuous phase to a higher extent than that in larger droplets. Upon diffusion along the concentration gradient through the continuous phase, the dissolved material comes across bigger droplets and redeposits upon them. As a result, the average particle size increases while the distribution width narrows. Since Ostwald ripening proceeds via molecular diffusion of dissolved lipid through the continuous phase, the droplets do not have to be close to each other for Ostwald ripening to occur. The rate of Ostwald ripening will increase as interfacial tension, the diffusion coefficient of the dissolved molecules, and lipid solubility in the continuous phase increase.13 Taking the expected effects of heat treatment on the emulsion into consideration, both mechanisms for particle growth seem possible: During heat treatment, the particle collision rate and the kinetic energy increase, which should both facilitate coalescence. However, diffusivity of the dissolved lipid and lipid solubility in the continuous phase increase as well, which should in turn facilitate Ostwald ripening. A point of particular relevance is the behavior of the emulsifier pol188 during autoclaving: An increase in temperature makes the nonionic surfactant pol188 less hydrophilic,15 so an aqueous solution of the surfactant separates into two phases and becomes turbid at a certain temperature, which is defined as the cloud point. The cloud point of pol188 is above 100 °C.16 Temperatures above the cloud point might lead to dehydration of the emulsifier, followed by a breakdown of the film around the lipid droplets and coalescence of the droplets. Apart from this, an increase in temperature also results in the formation of multimolecular aggregates, i.e., micelles, from the previously individually dispersed molecules of pol188.15 While phospholipid-stabilized nanoemulsions for parenteral nutrition are routinely autoclaved and hardly change during the process,2,17 the stability of other colloidal carriers upon autoclaving appears to be influenced by the type of emulsifier and lipid: Addition of oleic acid as cosurfactant to nanoemulsions stabilized with phospholipids increased stability upon autoclaving, while the addition of Tween 80, polyoxyethylene 60 hydrogenated castor oil, and cholesterol caused a substantial increase in particle size upon autoclaving.18 Nanoemulsions stabilized solely with nonionic surfactants, i.e., Tween 80, Solutol H15, or Cremophor EL, showed an increase in droplet size upon autoclaving, while nanoemulsions stabilized with pol188 remained stable.19 Deliberately lowering the cloud point of pol188 by adding NaCl or butyl diglycol also led to particle growth upon autoclaving.20 Stability of pol188-stabilized emulsions upon autoclaving also depended on the type of lipid: Castor oil or its mixtures with medium chain triglycerides or soybean oil gave an appropriate stability, while the latter oils alone did not produce stable emulsions.

2. MATERIAL AND METHODS 2.1. Chemicals and Reagents. The triglycerides tricaprin (Dynasan 110), trilaurin (Dynasan 112), trimyristin (Dynasan 114), tripalmitin (Dynasan 116), and tristearin (Dynasan 118), all from Hüls/Condea/Sasol/Cremer, Witten, Germany, were kind gifts from the manufacturer as were poloxamer 188 and poloxamer 407 (Kolliphor P 188/407, BASF, Ludwigshafen, Germany). Medium chain triglycerides and sodium dodecyl sulfate (SDS) were from Caelo (Hilden, Germany). Sucrose laurate was from Mitsubishi-Kagaku Food Corporation (Tokyo, Japan); sodium azide, potassium chloride, and glycerol were obtained from Roth (Karlsruhe, Germany) as were all syringe filters. Water was bidistilled quality. 2.2. Preparation of Lipid Nanodispersions and Heat Treatment. 10% of the respective lipid formed the lipid phase; the aqueous phase consisted of 2.25% glycerol, 0.05% 3112

DOI: 10.1021/acs.molpharmaceut.8b00202 Mol. Pharmaceutics 2018, 15, 3111−3120

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Molecular Pharmaceutics Table 1. Overview of Experiments and Dispersions experiment on effect of temperature emulsifier content

fatty acid chain length

fatty acid chain length combined with temperature

addition of KCl

other emulsifiers

lipid typea

z-average [nm]

PdI

emulsifier content [%]b

trimyristin C14 trimyristin C14 trimyristin C14 trimyristin C14 trimyristin C14 medium chain triglycerides C8 tricaprin C10 trilaurin C12 trimyristin C14 tripalmitin C16 tristearin C18 tricaprin C10 trilaurin C12 tripalmitin C16 tristearin C18 tricaprin C10 trilaurin C12 trimyristin C14 trimyristin C14 trimyristin C14

82 122 100 68 82 83 80 93 138 122 134 80 93 59 59 80 93 105 70 155

0.154 0.101 0.095 0.124 0.154 0.108 0.146 0.140 0.114 0.167 0.138 0.146 0.140 0.138 0.136 0.146 0.140 0.120 0.073 0.095

5 2.5 5 15 5 5 5 5 5 5 5 5 5 12 12 5 5 5 5 5% poloxamer 407

a

The lipids used are triacylglycerols (triglycerides) with different lengths of the esterified fatty acid chains. The number of carbon atoms of the fatty acids in the respective triglyceride are indicated by “C10” to “C18”. While all other triglycerides mainly contain the fatty acid residue of the indicated length (monoacyl triglycerides), medium chain triglycerides are a liquid mixture of mainly caprylic (C8) and capric acid (C10) triglycerides (indicated here as “C8”). bIf not stated otherwise, the emulsifier is pol188.

Following an equilibration time of 300 s, four measurements of 300 s each were run at 25 °C. z-Average and PdI were calculated as means of the four runs ± standard deviation. In addition, laser diffractometry with polarization intensity differential scattering (LD/PIDS) was performed (Beckman Coulter LS 13 320 (Beckman Coulter, Krefeld, Germany) with every sample to check for bimodal particle size distributions, especially after heat treatment. Three consecutive measurements of 90 s each were run to calculate the volume distribution of each sample by a Mie theory-based evaluation model: For the particles, a refractive index (RI) of 1.46 (i.e., in the center of the range of representative triglyceride oils: medium chain triglycerides: 1.44; soybean oil: 1.4830) and an absorption index of 0.01 (following the recommendation of the instrument’s manufacturer for translucent materials) was assumed, and for the aqueous dispersion medium, an RI of 1.333 was used. LD/PIDS results are only given when they show a bimodal distribution or entirely disagree with PCS results. LD/PIDS has been identified as an inappropriate measurement technique to reliably detect changes in the particle size distribution width of nanoemulsions like the ones used in this study.12 2.4. Differential Scanning Calorimetry. Differential scanning calorimetry (DSC) measurements were carried out using a Mettler Toledo DSC 1 STARe system with FRS 5 sensor, which had been calibrated with zinc and indium. Approximately 15 μg of emulsion was weighed into 40 μL aluminum pans (Mettler Toledo, Gießen, Germany) and cold welded. An empty pan was used as the reference. Samples were first heated to 80 °C, then cooled to −5 °C (thereby crystallizing the trimyristin emulsion droplets), and last, the particles were melted by heating to 80 °C, using a scan rate of 2.5 K/min. 2.5. Variations of Poloxamer Content, Lipid Content, Cloud Point, and Emulsifier. The content of pol188 was

sodium azide, and 2.5, 5, 12, or 15% pol188 or 5% poloxamer 407 dissolved in bidistilled water (the content of all ingredients is given related to the total starting weight of the emulsions (w/w)). Detailed information on the dispersion compositions can be found in Table 1. Both phases were mixed for 5 min with 15 000−19 000 rpm (T25 digital Ultra Turrax, IKA, Staufen, Germany), and the resulting pre-emulsion was then submitted to high-pressure homogenization. High-pressure homogenization (Microfluidizer M110-PS, interaction chamber type F12Y DIXC, Microfluidics, USA) was performed at 1500 bar (nanodispersions with 12% pol188) or 800 bar (all other dispersions). Lipids not liquid at room temperature were processed at 10 °C above their melting point, Tm (trimyristin Tm = 56 °C; tripalmitin Tm = 66 °C; tristearin Tm = 73 °C). Directly after homogenization, all dispersions were filtered (0.45 μm, polyvinylidene fluoride or 0.22 μm, polyether sulfone) and stored in glass vials (Zscheile & Klinger, Hamburg, Germany) at 20 °C. For heat treatment, the dispersions were aliquoted into 2 mL glass vials type 1 (Zscheile u. Klinger), sealed by chlorobutyl stoppers and crimped. Heat treatment at 101 °C and above was performed in an autoclave (Sanoclav TKL MCS 53, Adolf Wolf), while for heat treatment below 100 °C, the vials were immersed in a temperature-controlled water bath. The specified durations represent the effective heat treatment time at the specific temperature and not the entire process time. 2.3. Particle Size Analysis. The investigation of the intensity weighted mean diameter (z-average diameter) and polydispersity index (PdI) was accomplished by photon correlation spectroscopy (PCS) using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) at an angle of 173°. Prior to the measurements, each sample was diluted with purified and filtered water to obtain an appropriate scattering intensity (indicated by an automatically adjusted attenuator index of 7). 3113


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(Table 1) was autoclaved for 15 min at either 101, 110, 121, or 130 °C and subjected to PCS measurements (Figure 2A). The results showed that autoclaving at 101 °C had no effect, and heat treatment at 110 °C caused a decrease in PdI only but no substantial growth in particle size. As expected, autoclaving at 121 °C led to a decrease in particle distribution width and an increase in particle size. A temperature of 130 °C appeared to be too hot to improve the quality of the emulsion: Both PdI and particle size increased drastically. Studies on the effect of autoclaving duration at 121 °C revealed that 5 min were enough to decrease the particle size distribution width and to increase the particle size (Figure 2B). When the heat treatment was continued for 45 min or longer, the emulsion became less homogeneous as indicated by an increasing PdI (data not shown). This is in good agreement with experiments on the effect of multiple autoclaving (Supporting Information, Figure S1): After the first autoclaving process, each additional run at 121 °C slightly increased both particle size and PdI. After five runs, a bimodal particle size distribution was detected by LD/ PIDS measurements. 3.3. Effect of Emulsifier Content. To assess the effect of the emulsifier concentration during heat treatment, trimyristin nanoemulsions with 2.5, 5, or 15% pol188 were prepared. The three resulting nanoemulsions differed in initial particle size and particle size distribution width: The higher the pol188 content during production, the smaller the resulting particles (Figure 3A). The presence of small particles of different sizes in the samples prepared with 5 and 15% pol188 was

also varied after production of the nanodispersions to investigate its effect unbiased by different initial particle sizes of the nanoemulsion. For this, the amount of free pol188 in the nanodispersion was adjusted to about 1% pol188 by dialysis: 10 mL of nanodispersion was filled in a dialysis tube (Biotech CE Dialysis Tubing, Spectrum Laboratories, Frankfurt, Germany; cellulose ester, 100 kDa molecular weight cutoff) and immersed in 2 L of a solution of 1% pol188 and 2.25% glycerol under mild agitation for 65 h to reach equilibrium. After dialysis, refractive index measurements (refractometer Abbemat-WR, Anton Paar, Ostfildern-Scharnhausen, Germany) verified that the content of pol188 in the surrounding solution was 1.10%. The dialyzed nanodispersion was later also mixed with 25% pol188 solution to adjust higher pol188 contents and diluted with the dialysis medium to assess the effect of lipid content on the changes during heat treatment. The cloud points of solutions with 1, 5, and 12% pol188 in the presence of 0.25, 0.5, 1, 1.5, 2, and 5 M potassium chloride (KCl) were determined by gradually heating the solutions in a water bath. The cloud point was defined as the temperature at which the complete sample became turbid for the first time. The measured cloud points were plotted against the concentration of KCl, the data were combined in a linear equation, and the y-intercept, i.e., the cloud point at zero KCl, was defined as the cloud point of pol188. The function obtained was also used to deliberately decrease the cloud point of pol188 during heat treatment. The amount of KCl necessary to adjust a certain cloud point was derived from the linear function, and the cloud point of pol188 in nanoemulsions of C10, C12, and C14 triglycerides was adjusted to defined values by adding the respective amount of KCl. The nanoemulsion was then subjected to heat treatment 10 °C above the adjusted cloud point. In order to study emulsifier effects, 5 or 10% of pol188 or SDS was added to a trimyristin nanoemulsion stabilized with 5% pol188 prior to autoclaving. In addition, nanoemulsions prepared with poloxamer 407 or sucrose laurate as emulsifier only were subjected to heat treatment.

3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of Lipid Nanodispersions. Depending on the sample composition and the homogenization conditions, triglyceride nanodispersions with mean particle sizes between about 60 and 155 nm were produced (Table 1). The PdI was always below 0.2. Standard deviations between the four measurement runs were generally small (less than 1 nm in z-average diameter and less than 0.01 in PdI in more than 90% of all samples) and were not included for reasons of readability. Trimyristin and other monoacidic triglycerides exhibit strong supercooling in the nanodispersed state. So in contrast to tripalmitin and tristearin nanoparticles, trimyristin droplets as prepared here remain liquid at room temperature even though trimyristin melts around 56−57 °C.31 For tricaprin, trilaurin, and trimyristin, the liquid state of the droplets was checked by differential scanning calorimetry. Heating these dispersions from 20 to 80 °C yielded no melting event, which confirmed that the particles remained in a supercooled liquid state. Dispersions of tripalmitin and tristearin yielded a melting event upon heating, which verified their solid state at room temperature. 3.2. Effect of Heat Treatment Temperature and Duration. An emulsion with 10% trimyristin and 5% pol188

Figure 2. Effect of temperature and duration of heat treatment on the change in particle size (z-average) and particle size distribution width (PdI) of a trimyristin nanoemulsion with 5% pol188. (A) Heat treatment at different temperatures for 15 min. (B) Heat treatment at 121 °C for different durations. 3114


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there should be hardly any free pol188 in the emulsion sample prepared with 2.5% pol188. In contrast to that, the distribution width decreased drastically in the emulsion prepared with 15% PdI (Figure 3A,B). Overall, the results indicated that at least some free pol188 was necessary to induce the desired effect. Since dissimilarities in the nanoemulsion composition before autoclaving confounded the interpretation, a consecutive experiment was performed where the concentration of pol188 was varied after emulsion preparation: In an emulsion prepared with 5% pol188 and 10% lipid, the content of free emulsifier in the continuous phase of the nanoemulsion was adjusted to 1% via dialysis. This left particle size and particle size distribution width unchanged as confirmed by PCS (data not shown). This dialyzed emulsion with 1% free pol188 was autoclaved and analyzed (Figure 4). Also, the emulsion was mixed with concentrated pol188 solution to increase the content of free pol188 to 15%. Both samples showed a strong decrease in PdI upon autoclaving; only the increase in particle size was slightly less in the sample with 1% free pol188. So the effect of different emulsifier contents after emulsion preparation was comparatively small, since the decrease in PdI was the same in all three samples and only the increase in particle size varied slightly. The nanoemulsion dialyzed to 1% free pol188 was diluted with a 1% pol188 solution to investigate the effect of lipid content during heat treatment (Figure 4, right). The PdI decreased in all samples but the sample with 1% lipid, which was considered an outlier. A decrease in lipid content led to a slightly bigger increase in particle size. To sum up, a small amount of free pol188 seemed to be crucial for the desired effect, whereas the lipid content had only a little effect on the change in PdI and particle size. This was further supported in an additional experiment: A nanoemulsion (10% trimyristin, 5% pol188) was diluted with water to different concentrations of both trimyristin and pol188. This way, the ratio of trimyristin to pol188 remained unchanged, the lipid content decreased, and some free pol188 remained. The changes upon autoclaving were comparable with respect to particle size in all five samples (Supporting Information, Figure S2). In contrast to the situation with poloxamer 407-stabilized monoolein

Figure 3. Effect of the content of pol188 at emulsion preparation on the native and autoclaved trimyristin emulsion (121 °C, 15 min) (A) Effect on particle size (z-average) and particle size distribution width (PdI). (B) Effect on the melting event. The melting event was analyzed via differential scanning calorimetry (DSC). Beforehand, the initially liquid trimyristin droplets had been crystallized by cooling to −5 °C in the DSC.

particularly noticeable in the DSC thermograms (Figure 3B). Crystalline trimyristin nanoparticles display a size-dependent melting behavior.32,33 Smaller and consequently thinner particles melt at lower temperatures than bigger, thicker crystals, which melt almost at the same temperature as bulk trimyristin, i.e., 56−57 °C. Consequently, small nanoparticles of slightly different sizes in the same nanodispersion result in a melting event with distinct, separate transitions. Thus, a single, sharp melting event indicates nanoparticles of similar size, whereas a broad, jagged peak points to small nanoparticles of different sizes. The irregular melting peaks of the nonautoclaved particles prepared with 5 and 15% pol188 indicated the presence of small particles of different size. The melting peak of the recrystallized trimyristin nanoparticles prepared with 2.5% was comparatively sharp and uniform, which pointed to a narrower particle size distribution. Upon autoclaving, the small particles with 15% pol188 grew considerably, whereas the particles prepared with 2.5% pol188 became hardly any bigger (Figure 3A). Since the particles were of different size before autoclaving, the observed difference in particle growth upon autoclaving could very well be biased toward a strong effect of pol188 content. However, the change in particle distribution width showed similar trends: For emulsions with 2.5% pol188, the change in distribution width was only small. In our previous study,12 we showed that 28 mg of pol188 was necessary to stabilize 100 mg of trimyristin dispersed into 140 nm large emulsion droplets. Consequently,

Figure 4. Effect of the content of emulsifier and lipid during heat treatment. The first, white bar refers to the emulsion prior to autoclaving; all other bars give the data after autoclaving. Left: Via dialysis, the content of free pol188 in a trimyristin nanoemulsion prepared with 10% lipid and 5% pol was adjusted to 1%. Dilution with concentrated pol188 solution led to 15% free pol188, so the lipid content was simultaneously reduced. Right: Effect of lipid content at a constant free pol188 concentration of 1%. 3115


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Molecular Pharmaceutics dispersions,22 differences in lipidor lipid and emulsifier content can thus not be utilized to achieve deliberate alterations in particle size of pol188-containing trimyristin dispersions. 3.4. Effect of Lipid Type. Six triglycerides, which varied in esterified fatty acid chain length from 8 to 18 carbon atoms, were processed to nanodispersions. The melting points and lipophilicity of the lipids increased with increasing fatty acid chain length. The solid state of nanoparticles from tripalmitin and tristearin (C16 and C18) at room temperature had been verified by DSC measurements. Differences in viscosity and lipophilicity of the triglycerides as well as the solid state of C16 and C18 particles resulted in different initial particle sizes of the nanodispersions (Table 1). The effect of autoclaving (121 °C, 15 min) on the nanodispersions depended strongly on lipid type (Figure 5A). Nanoemulsions of the rather polar lipids tricaprylin (C8) and tricaprin (C10) showed particles in the micrometer range and a bimodal distribution (LD/PIDS) after autoclaving. Trilaurin displayed strong, but not excessive, particle growth and a strong decrease in PdI. For trimyristin, the particle growth was moderate, and the decrease in PdI was strong. Tripalmitin particles grew slightly, and the PdI decreased noticeably, while hardly any effect could be observed for the tristearin particles. For the lipids trimyristin (C14), tripalmitin (C16), and tristearin (C18), also the melting event of the crystalline particles could be analyzed via DSC (Figure 5B). In contrast to C14 nanoemulsions, particles from C8,

C10, and C12 triglycerides could not be crystallized by cooling to −5 °C and could consequently not be analyzed by DSC. The untreated dispersions of C14, C16, and C18 triglycerides produced irregular, broad, and jagged melting events, which resulted from the separate melting of particle fractions of different size. After autoclaving, particles of trimyristin (C14) melted in one sharp peak, indicating that the particles were now of similar size. C16 and C18 particles still showed a stepwise melting behavior after autoclaving, which pointed to the presence of particle fractions of different size. The results of DSC measurements were thus in good agreement with the PCS results. Overall, the change in particle size and particle size distribution width upon autoclaving appeared to be strongly influenced by the nature, i.e., the polarity, of the lipid. This was further supported by similar studies with C18 triglyceride dispersions, which differed in polarity due to different degrees of saturation and number of hydroxyl groups (Supporting Information, Figure S3). As lipid polarity increased, so did the particle size growth and the effect on PdI upon autoclaving. To sum up, the interim results indicated that the desired changes upon autoclaving strongly depended on the temperature, were heavily influenced by the lipid type, and required at least some free pol188 to occur. On the basis of these results, two temperature-sensitive mechanisms were conceivable: First, the solubility of the lipid had to reach a certain range, which was exceeded for polar lipids like C8 and C10 triglycerides and was hardly or not all reached even at 121 °C for nonpolar lipids like C16 or C18 triglycerides. Second, it was also possible that the change in temperature influenced the behavior of the emulsifier pol188, e.g., induced the formation of micelles or decreased pol188 solubility by exceeding its cloud point. To study the effects in more detail, dispersions from lipids of different polarity were subjected to different temperatures. 3.5. Combined Effect of Lipid Type and Temperature. Dispersions of C10, C12, C16, and C18 triglycerides were treated at different temperatures. Dispersions of C16 and C18 triglycerides were produced with 12% pol188 at high pressures to obtain small particles, which would hopefully make it easier to detect changes upon heat treatment (Table 1). Of note, in the untreated dispersion of C10 triglycerides, the PdI was already rather low, so only a small decrease in PdI could be expected. Depending on the polarity of the lipid, the desired effect was found at different temperatures (Figure 6). C10 triglyceride particles grew considerably at 110 °C, and for C12 triglyceride dispersions, a slight increase in particle size combined with a decrease in PdI also occurred at 110 °C. Even though the content of pol188 was 12% and the particles were very small, the nonpolar triglycerides of C16 and C18 fatty acids showed only small effects on particle growth at 121 °C but grew in particle size at 130 °C. Changes in PdI could be observed already at 121 °C. DSC analysis of the melting events verified that 130 °C was necessary to achieve a sharp, defined melting peak for nanoparticles of these two triglycerides (Supporting Information, Figure S4). These results pointed to an influence of lipid solubility in the water phase of the dispersion: Triglycerides with short esterified fatty acids are more polar and may reach sufficient water solubility at comparatively low temperatures, whereas the very lipophilic lipids from C16 and C18 fatty acids require higher temperatures to attain sufficient water solubility. To assess the influence of temperature-

Figure 5. (A) Effect of fatty acid chain length of saturated triglycerides on effect of heat treatment at 121 °C for 15 min. (A) Particle size (z-average) and particle size distribution width (PdI) of the native and autoclaved dispersions. (B) Effect on the melting event of the native and autoclaved dispersions for C14, C16, and C18 triglycerides. The melting events were analyzed via DSC. Beforehand, the initially liquid C14 triglyceride droplets had been crystallized by cooling to −5 °C in the DSC. C8, C10, and C12 triglycerides in the nanodispersed state could not be crystallized by cooling to −5 °C. 3116


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this setup, the C10 triglyceride, the particle size doubled at 110 °C. However, with KCl present, the particle size grew much more, i.e., to 450 nm, at the same temperature. At 95 and 85 °C, particle growth without KCl was very limited, while the particle size increased substantially at the same temperatures when KCl had been added to the nanoemulsions. At 60 °C, the emulsion remained unchanged with and without KCl. From these results, two conclusions could be drawn: First, exceeding only the cloud point was not sufficient to induce particle growth. Second, KCl had a strong influence on particle growth, but only once a certain temperature was reached. This temperature was, however, not related to the cloud point of pol188 in the respective emulsion. The results for C12 and C14 triglycerides confirmed these conclusions drawn from C10 triglyceride particles, even though the increase in particle size was in general smaller in these two dispersions. This mild change in particle size was due to the decreasing lipid polarity from C10 to C14 triglycerides as already outlined in section 3.4 In the dispersions of C12 and C14 triglycerides, the influence of KCl is easiest to discern in the change of PdI: For the C12 triglyceride, the PdIas well as the particle size remained unchanged with and without KCl at 60 °C. At 85 °C, however, the PdI was considerably reduced in the presence of KCl. The particle size, however, hardly changed at 85 °C with and without KCl, as the lipid is less polar than the C10 triglyceride. For the C12 triglyceride, 110 °C and the presence of KCl were necessary to increase the particle size, but changes in PdI could already be seen at 85 °C. For the C14 triglyceride, 100 °C was necessary to induce a decrease in PdI in the presence of KCl. Consequently, exceeding the cloud point was not the decisive factor in particle growth, and the addition of KCl had a strong effect on particle growth only once a certain temperature was reached. The less polar the dispersed triglyceride was, the higher was the temperature that was necessary to induce particle growth. 3.7. Effect of Other Emulsifiers. The previous experiments did not support a strong effect of the emulsifier’s cloud point, even though the addition of KCl did influence the process. To narrow down the essential characteristics of the emulsifier, the influences of different emulsifiers during heat treatment was tested: SDS as an anionic emulsifier should be able to prevent coalescence of droplets also at high temperatures. Poloxamer 407, which was also used in the studies by Barauskas et al., has a much lower critical micellization concentration than pol188 but also a cloud point above 100 °C.15,34 Sucrose laurate is an nonionic emulsifier with no cloud point and a critical micellization concentration below 0.5 mM.35 If the cloud point of the emulsifier was essential, emulsions stabilized with sucrose laurate should be stable upon autoclaving. Addition of 5 or 10% pol188 to a trimyristin nanoemulsion stabilized with 5% pol188 led to slight increase in particle growth and a decrease in PdI (Figure 8), similar to the results given in Figures 3 and 4. Addition of 5 or 10% of the anionic emulsifier SDS caused stronger particle growth but led to an increase in PdI at the same time. LD-PIDS measurements underlined that the particle size distribution width had become wider in samples with SDS (Supporting Information, Figure S6). A trimyristin nanoemulsion prepared with 5% poloxamer 407 basically retained its particle size upon autoclaving while its PdI decreased. In contrast to that, autoclaving a trimyristin nanoemulsion (5% lipid) stabilized with 6% sucrose laurate led

Figure 6. Combined effect of fatty acid chain length of saturated triglycerides in combination with heating to different temperatures for 15 min on particle size (z-average) and particle size distribution width (PdI) of the dispersions. Please note the different temperature regimes for the dispersions of C10 and C12 triglycerides (left) and dispersions of C16 and C18 triglyceride (right). Dispersions of C16 and C18 triglycerides were produced with 12% pol188.

dependent behavior of the emulsifier pol188 as well, its cloud point was defined and adjusted in the next set of experiments. 3.6. Variations of Emulsifier Cloud Point. Solutions with 1, 5, and 12% pol188 and different amounts of KCl were slowly heated to determine the respective cloud point (Supporting Information, Figure S5). The y-intercept gave the cloud point at 0 mol/l KCl, i.e., the cloud point of pol188 as 103.2 °C. On the basis of the equation from this plot (Figure S5), the cloud point of pol188 in nanoemulsions of C10, C12, and C14 triglycerides was adjusted to defined values. The nanoemulsion was then subjected to heat treatment 10 °C above the adjusted cloud point, so irrespective of the applied temperature, the cloud point of pol188 was exceeded in every setup by 10 °C. Particle size and PdI after heat treatment of the different emulsions are depicted in Figure 7. For the most polar lipid in

Figure 7. Effect of the addition of KCl before heat treatment (15 min) on particle size (z-average) and particle size distribution width (PdI) of C10, C12, and C14 triglyceride emulsions. The amount of KCl added was calculated to lower the cloud point of the emulsifier pol188 to 10 °C below the respective heat treatment temperature. For example, the sample “+ KCl 60°C” contained just enough KCl to reduce the cloud point to 50 °C. This sample was then treated at 60 °C, i.e., 10 °C above the adjusted cloud point. Patterned bars mark the minimum temperature necessary to observe effects of KCl in the respective triglyceride emulsion. 3117


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pol188 had been reached during autoclaving. Consequently, the emulsifier’s cloud point was only of minor impact. Addition of KCl to the continuous phase does not only lower the cloud point but also the critical micellization concentration or the critical micellization temperature.36 Accordingly, the addition of KCl could lower the critical micellization temperature for pol188 and thus amplify the formation of micelles from free pol188. Mass transfer in Ostwald ripening proceeds predominantly via molecular diffusion across the continuous phase, but micelles of nonionic surfactants can accelerate the rate of Ostwald ripening up to a factor of 50.23−25,27 The effect of KCl on particle growth and decrease in PdI during heat treatment might thus result from an increased Ostwald ripening rate because of increased micellar transport. The effect of the amount of free pol188 in the dispersion can be plausibly described by the same mechanism: elevated temperatures induce the formation of micelles, which in turn increase the rate of Ostwald ripening. If the amount of free pol188 is very limited or zero, lipid transport via micelles cannot contribute to Ostwald ripening. Larger amounts of free pol188 will in turn increase the rate of Ostwald ripening. If the predominant destabilizing mechanism was coalescence, the addition of the anionic surfactant SDS should minimize particle growth during autoclaving. However, the addition of SDS did not improve but worsen emulsion stability. Since sucrose laurate does not contain polyoxyethylene groups, distinct alterations in its interaction with water upon increasing temperatures (like clouding or phase separation) are not expected. Still, the emulsion stabilized with sucrose laurate broke during autoclaving while the emulsion stabilized with poloxamer 407 (cloud point above 100 °C) remained stable. So the emulsifier plays a crucial role during heat treatment, but this influence should not be attributed solely to the emulsifier’s cloud point. In previous studies on the effect of autoclaving on lipid nanocarriers, the authors proposed fusion, flocculation, or coalescence as the destabilizing mechanism.18,19,22 However, the obtained results could also be explained by Ostwald ripening: Emulsions stabilized with phospholipids remain stable during autoclaving. Phospholipids do not form micelles but vesicles, which rules out micelle-enhanced Ostwald ripening for phospholipids. This theory is supported by a finding of Groves and Herman,37 who discovered that during sterilization, surplus phospholipids from the aqueous phase relocated permanently to the oil phase of the emulsion. The finding that the addition of cosurfactants induced particle growth during autoclaving in nanoemulsions stabilized with phospholipids could be explained by the formation of cosurfactant micelles and accelerated Ostwald ripening.18 Also, the strong influence of the lipid matrix described by Jumaa et al.19 might result from different lipid polarities rather than differences in surface tension of the lipids. The destructive effect of NaCl during autoclaving20,22 might also be explained by the amplified formation of micelles from pol188 and poloxamer 407 in the presence of chloride. The finding that the particle size of poloxamer 407-stabilized monoolein dispersions upon autoclaving increased almost linearly with an increase in amphiphile content might also be explained by micelle formation: The amphiphile content was between 1 and 5% monoolein and poloxamer 407 in the dispersion. Since the ratio of lipid to emulsifier was 9:1, the samples contained 0.1 to 0.5% poloxamer 407. Given that the critical micellization concentration at 40 °C is 0.005%,34 small differences of the concentration in this range might have a large influence on

Figure 8. Left: Effect of the addition of pol188 or SDS before autoclaving (15 min, 121 °C) on particle size (z-average) and particle size distribution width (PdI) of a C14 triglyceride emulsion prepared with 5% pol188. Right: Effect of autoclaving (15 min, 121 °C) on particle size (z-average) and particle size distribution width (PdI) of a C14 triglyceride emulsion prepared with 5% poloxamer 407. White bars refer to the emulsions prior to autoclaving; all other bars give the data after autoclaving.

to a bimodal size distribution and particle sizes up to 50 μm (data not shown). 3.8. Concluding Discussion. The experiments revealed a strong effect of lipid polarity on particle growth upon heat treatment. This finding strongly pointed to a form of Ostwald ripening as the underlying mechanism of particle growth (Figure 9). A coalescence-based mechanism could not explain the strong influence of lipid polarity on the observed effects. The fact that dilution of the nanoemulsion slightly increased particle growth is also in good agreement with an Ostwald ripening mechanism, as this is not considered to be strongly volume-fraction-dependent.13 Coalescence, on the contrary, increases as the fraction of dispersed phase increases.14 So the effects of temperature and lipid polarity on the results after autoclaving could both be attributed to an increase in lipid solubility in the continuous phase. However, the effects of the amount of pol188 in the emulsion and of KCl still have to be considered: Addition of KCl decreases the cloud point of pol188. However, heat treatment upon addition of KCl showed that exceeding the cloud point was not sufficient to induce particle growth. Also, nanodispersions of C16 and C18 triglycerides hardly grew, even though the cloud point of

Figure 9. Schematic illustration of accelerated Ostewald ripening during heat treatment. More polar triglycerides have higher solubility in the aqueous phase and require a lower temperature for increased molecular diffusion to occur. A certain amount of free poloxamer is required for the process, probably since poloxamer micelles formed at elevated temperatures enhance lipid transport. 3118


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(4) Gaumet, M.; Vargas, A.; Gurny, R.; Delie, F. Nanoparticles for drug delivery: The need for precision in reporting particle size parameters. Eur. J. Pharm. Biopharm. 2008, 69, 1−9. (5) Kupetz, E.; Bunjes, H. Lipid nanoparticles: drug localization is substance-specific and achievable load depends on the size and physical state of the particles. J. Controlled Release 2014, 189, 54−64. (6) Göke, K.; Bunjes, H. Drug solubility in lipid nanocarriers: Influence of lipid matrix and available interfacial area. Int. J. Pharm. 2017, 529, 617−628. (7) Joseph, S.; Bunjes, H. Preparation of nanoemulsions and solid lipid nanoparticles by premix membrane emulsification. J. Pharm. Sci. 2012, 101, 2479−2489. (8) Mabille, C.; Schmitt, V.; Gorria, P.; Leal Calderon, F.; Faye, V.; Deminière, B.; Bibette, J. Rheological and shearing conditions for the preparation of monodisperse emulsions. Langmuir 2000, 16, 422− 429. (9) Anna, S. L.; Bontoux, N.; Stone, H. A. Formation of dispersions using “flow focusing” in microchannels. Appl. Phys. Lett. 2003, 82, 364−366. (10) Bibette, J. Depletion interactions and fractionated crystallization for polydisperse emulsion purification. J. Colloid Interface Sci. 1991, 147, 474−478. (11) Dutz, S.; Kuntsche, J.; Eberbeck, D.; Müller, R.; Zeisberger, M. Asymmetric flow field-flow fractionation of superferrimagnetic iron oxide multicore nanoparticles. Nanotechnology 2012, 23, 355701. (12) Göke, K.; Roese, E.; Arnold, A.; Kuntsche, J.; Bunjes, H. Control over particle size distribution by autoclaving poloxamerstabilized trimyristin nanodispersions. Mol. Pharmaceutics 2016, 13, 3187−3195. (13) Taylor, P. Ostwald ripening in emulsions. Adv. Colloid Interface Sci. 1998, 75, 107−163. (14) Tadros, T. F. Emulsions: Formation, stability, industrial applications; De Gruyter: Berlin, 2016. (15) Kabanov, A. V.; Nazarova, I. R.; Astafieva, I. V.; Batrakova, E. V.; Alakhov, V. Y.; Yaroslavov, A. A.; Kabanov, V. A. Micelle formation and solubilization of fluorescent probes in poly(oxyethylene-b-oxypropylene-b-oxyethylene) solutions. Macromolecules 1995, 28, 2303−2314. (16) BASF Nutrition and Health. Kolliphor® P Grades; 03_111136e-03; 2013; https://industries.basf.com/bin/bws/ documentDownload.en.8805243136469. (17) Lee, G.; Groves, M. J. A pragmatic approach to the particle sizing of submicrometre emulsions using a laser nephelometer. Powder Technol. 1981, 28, 49−54. (18) Yamaguchi, T.; Nishizaki, K.; Itai, S.; Hayashi, H.; Ohshima, H. Physicochemical characterization of parenteral lipid emulsion: Influence of cosurfactants on flocculation and coalescence. Pharm. Res. 1995, 12, 1273−1278. (19) Jumaa, M.; Müller, B. W. The stabilization of parenteral fat emulsion using non-ionic ABA copolymer surfactant. Int. J. Pharm. 1998, 174, 29−37. (20) Jumaa, M.; Kleinebudde, P.; Müller, B. W. Physicochemical properties and hemolytic effect of different lipid emulsion formulations using a mixture of emulsifiers. Pharm. Acta Helv. 1999, 73, 293−301. (21) Wörle, G.; Siekmann, B.; Koch, M. H. J.; Bunjes, H. Transformation of vesicular into cubic nanoparticles by autoclaving of aqueous monoolein/poloxamer dispersions. Eur. J. Pharm. Sci. 2006, 27, 44−53. (22) Barauskas, J.; Johnsson, M.; Joabsson, F.; Tiberg, F. Cubic phase nanoparticles (Cubosome): Principles for controlling size, structure, and stability. Langmuir 2005, 21, 2569−2577. (23) Binks, B. P.; Clint, J. H.; Fletcher, P. D. I.; Rippon, S.; Lubetkin, S. D.; Mulqueen, P. J. Kinetics of swelling of oil-in-water emulsions stabilized by different surfactants. Langmuir 1999, 15, 4495−4501. (24) Ariyaprakai, S.; Dungan, S. R. Influence of surfactant structure on the contribution of micelles to Ostwald ripening in oil-in-water emulsions. J. Colloid Interface Sci. 2010, 343, 102−108.

micelle concentration and also micelle-enhanced Ostwald ripening.

4. CONCLUSION Increase in particle size and decrease of particle size distribution width upon heat treatment of triglyceride dispersions stabilized with pol188 was found to proceed via an accelerated Ostwald ripening mechanism. While the fact that the small particles disappeared during this process already pointed to Ostwald ripening in the past, the strong influence of lipid polarity gave further evidence for this mechanism. Formation of micelles from the nonionic surfactant pol188 at elevated temperatures or in the presence of KCl presumably contributed to lipid transfer across the continuous phase. In contrast to moderate particle growth, emulsion breakup during autoclaving may very well proceed entirely via coalescence, or the droplets grow first via Ostwald ripening and coalesce once they reach a critical size. Because of the data presented here and the more detailed knowledge of the underlying mechanism, effective heat treatment is no longer limited to trimyristin nanoemulsions. Colloidal dispersions of various triglycerides can now be treated with heat to obtain a narrower particle size distribution.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.8b00202.

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Additional results on heat treatment experiments and cloud point determinations (PDF)

AUTHOR INFORMATION

Corresponding Author

*Tel.: +49 531 3915652; E-mail: [email protected]. ORCID

Katrin Göke: 0000-0002-5421-5731 Heike Bunjes: 0000-0002-8670-6076 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Miriam Wollbrink for carrying out much of the practical work and Nadine Francke for performing some follow-up experiments. K. Göke thanks the Niedersächsisches Ministerium für Wissenschaft und Kultur (MWK), Hannover, Germany for financial support in the joint research project SynFoBiA“Novel synthesis and formulation methods for poorly soluble drugs and sensitive biopharmaceuticals” of the Center of Pharmaceutical Engineering (PVZ).

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REFERENCES

(1) Bunjes, H. Lipid nanoparticles for the delivery of poorly watersoluble drugs. J. Pharm. Pharmacol. 2010, 62, 1637−1645. (2) Floyd, A. G. Top ten considerations in the development of parenteral emulsions. Pharm. Sci. Technol. Today 1999, 2, 134−143. (3) Maeda, H.; Nakamura, H.; Fang, J. The EPR effect for macromolecular drug delivery to solid tumors: Improvement of tumor uptake, lowering of systemic toxicity, and distinct tumor imaging in vivo. Adv. Drug Delivery Rev. 2013, 65, 71−79. 3119


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