1. Introduction
Most drug candidates are highly lipophilic in nature, making their high strength solubilization in liquid dosage forms a challenge. Additionally, most drugs are easily susceptible to chemical and/or enzymatic degradation, which reduces their bioavailability at the action site. To tackle these issues, several strategies have been developed, with the encapsulation of molecules in nanosystems being one of the most successful [
1,
2].
Nanosystems are typically classified as colloidal structures with a size of less than 500 nm, inside which drugs are incorporated. This will allow not only to increase the preparation’s drug strength, but also to protect the molecules from degradation, increase their permeability through biological membranes, promote bioavailability, reduce plasma protein binding, and allow a controlled drug release, with the possibility of targeting certain organs. There are many types of nanosystems, polymeric and lipid nanosystems being the most frequently used. There is a great variety of lipid nanosystems: liposomes and derived structures (transfersomes, niosomes, cubosomes, ethosomes, etc.), solid lipid nanoparticles, nanostructured lipid carriers, and nanometric emulsions [
3,
4]. Nevertheless, many of these nanocarriers have important disadvantages, such as being complex, time-consuming and having polluting preparation methods, low physical stability, low encapsulation efficiency, and lack of biocompatibility. However, these disadvantages are reduced or simply do not apply to nanometric emulsions, particularly those with the right formulas, which have good stability, solubilization capacity, and simple preparation methods.
Nanometric emulsions include nanoemulsions (NE) and microemulsions. Microemulsions, named as such since 1959 [
5], are thermodynamically stable and spontaneously form liquid dispersions of lipids and water, generally implying high amounts of surfactants and co-solvents/co-surfactants [
6,
7]. The droplet size is usually inferior to 100 nm, often not much larger than simple surfactant micelles, in which case they may appear completely transparent. The nature of their composition makes them related to classic emulsions, which are, however, biphasic thermodynamically unstable systems.
In turn, NE are colloidal liquid-in-liquid dispersions, usually presenting a size below 200 nm and a higher kinetic stability than macroemulsions. Compared with microemulsions, NE are usually composed of a lower content of hydrophilic surfactants and co-solvents, and usually require high energy methods for shearing and homogenizing the lipid droplets. Nevertheless, specific NE compositions can also be prepared by low energy methods, but with some compromise in the amounts of surfactant and co-solvents that are used (which tends to be high, although not as much as in microemulsions), and in the degree of homogeneity, which is tendentially low [
8].
Oil-in-water NE are a good strategy to deliver solubilized lipophilic drugs at high drug strengths. This is of high importance, especially for some delivery routes, such as in intranasal administration to the systemic circulation or to the brain, given the relatively short residence time and small volume of administration [
9,
10,
11,
12].
Hence, the purpose of this work was to develop a NE using low amounts of surfactants and/or cosolvents, thus with high lipid content and good solubilization of lipophilic drugs like simvastatin, while able to promote the absorption of drugs with low permeability like fosphenytoin. At the same time, it was aimed at a homogenous size under 200 nm and, preferably, a low energy preparation method. We screened NE compositions with a high proportion of relatively polar lipids aiming to achieve a good solubilization of drugs such as simvastatin and then characterized their droplet size. A composition with low proportion of hydrophilic surfactant to oils and no co-solvent originated a NE about 100 nm in droplet size using a simple low energy phase inversion method. Surprisingly, these NE spontaneously became highly homogenous upon refrigeration, with a polydispersity index (PDI) < 0.1, which is not usually found in NE without homogenization. Therefore, we secondarily aimed to establish which formulation factors were required for this attribute. The extremely low PDI was lost when changing excipients to similar ones or by changes in their proportion outside a narrow range. We also describe alternative compositions of the aqueous phase in which the NE display PDI < 0.1 already at room temperature. Given the high lipid content of such NEs, a high drug strength of lipophilic substances was obtained. Furthermore, the intranasal administration of the NE of a hydrophilic prodrug (fosphenytoin) to mice demonstrated a faster absorption than an aqueous solution of the drug.
2. Materials and Methods
2.1. Materials and Reagents
The hydrophilic surfactants Kolliphor® RH 40 (Macrogolglycerol hydroxy stearate), Kolliphor® EL (Macrogolglycerol ricinoleate), Kolliphor® P124 (Poloxamer 124), Kolliphor® HS 15 (Polyethylene glycol 660 12-hydoxystearate), and the oil Kollicream® IPM (Isopropyl Myristate) were kindly offered by BASF (Ludwigshafen, Germany); Transcutol® HP (Diethylene glycol monoethyl ether), a cosolvent, Labrasol® ALF (Caprylocaproyl polyoxyl-8 glycerides), a hydrophilic surfactant, and the oils Capryol® 90 (Propylene Glycol Monocaprylate (type II) NF), Capryol® PGMC (Propylene glycol monocaprilate, Type I), LabrafacTM PG (Propylene glycol dicaprylocaprate), Maisine® CC (Glycerol monolinoleate), and PeceolTM (Glycerol mono-oleate) were kindly offered by Gattefossé (Saint-Priest, France); the oils Imwitor® 948 (Glyceryl mono-oleate), Imwitor® 988 (Glycerol monocaprilate, Type I), and Softisan® 64S (Bis-diglyceryl polyacyladipate-2) were kindly offered by IOI Oleo GmbH (Hamburg, Germany); Capmul® MCM (Glycerol monocaprilocaprate), Capmul® 808G EP/NF (Glycerol monocaprylate Type II), Capmul® PG-8 (Propylene glycol monocaprylate), and Capmul® PG-8-70 NF (Propylene glycol monocaprylate Type II) were kindly offered by Abitec; Miglyol® 812 (medium-chain triglycerides; Caprylic/Capric Triglyceride), Soybean oil, Span® 80 (Sorbitane mono-oleate), Vitamin E Acetate, Cetiol V (Decyl oleate), the hydrophilic surfactants Tween® 20 (Polysorbate 20) and Tween® 80 (polysorbate 80), and the polymers Polyethylene glycol (PEG) 4000, (Hydroxypropyl)methyl cellulose (HPMC, corresponding to Hypromellose Viscosity 4000 mPa·s), and Polyvinylpyrrolidone (PVP, corresponding to Povidone K30) were acquired from Acofarma® (Madrid, Spain); the surfactant Tyloxapol was from Acros Organics (Thermo Fisher ScientificGeel, Belgium); Malic acid was acquired from Applichem (Darmstadt, Germany). Ultra-pure water was obtained from a Mili-Q® purification system from Millipore (Billerica, MA, USA). Simvastatin (98.03% purity) was purchased from Bld Pharmatech GmbH. (Kaiserslautern, Germany.) and kept at 4 °C under a nitrogen atmosphere during utilization. The bovine serum albumin (BSA) was acquired from Sigma-Aldrich, Inc (St. Louis, MO, USA). Acetonitrile and methanol were high-performance liquid chromatography (HPLC) gradient grade. The symbols TM and® will be omitted from now on for simplification.
2.2. Nanoemulsions Preparation
The oil phase (preconcentrate) was prepared by weighing the lipids and surfactants and mixing them from a few seconds to a few minutes until a homogenous solution was obtained. Depending on the NE, the aqueous phase either consisted in water, a 30 mM pH 5 malate buffer, or a 20 mM pH 7 phosphate buffer, to which NaCl, BSA, PEG 4000, HPMC, or PVP were added at the indicated concentrations (presented with the respective data in the results section). To prepare the NE, a phase inversion method was used, in which about a quarter of the final aqueous phase mass was first added and mixed with e preconcentrate, followed by the addition and mixture of the remaining aqueous phase mass.
2.3. Nanoemulsion’s Droplet Size and Zeta Potential
Both hydrodynamic diameter (droplet size) and PDI were measured by dynamic light scattering (DLS) technique associated with cumulants analysis. For that, a Zetasizer Nano ZS (Malvern®, United Kingdom) combined with the Zetasizer software (version 7.10) was used. Before each measurement, samples were diluted, about 500-fold, in ultra-pure water. For each tested formulation, two independent discardable cuvettes were prepared and three different measurements of each were automatically performed by the equipment set either at 20 or 25 °C. Measurements were typically performed within 30 min after dilution. As measurement parameters set in Zetasizer software, water was considered as the dispersant (Refractive Index = 1.330 and Viscosity = 0.8872 cP) and the material was set to a Refractive Index = 1.450, representing “lipid”). When assessing the role of refrigeration in mean droplet size and PDI, formulations were placed at 4 °C overnight (at least 12 h), and then measurements were performed exactly as stated before, after diluting the samples immediately upon removing them from the refrigerator.
Zeta potential was measured with Malvern’s Dip Cell Kit in the same equipment, using the same preparation steps. Measurements were taken at 20 or 25 °C and water was selected as the dispersant (Dielectric Constant = 78.5) and “lipid” as the material.
2.4. Osmolality
Osmolality was measured using Osmomat® 3000 freezing point osmometer from Gonotec® GmbH (Berlin, Germany). Osmolality measurement was performed in independent triplicates. The device was previously calibrated using ultra-pure water and two standard solutions of 300 mOsmol/Kg and 850 mOsmol/Kg.
2.5. Viscosity
Viscosity measurements were performed at different rotational velocities in a Brookfield DV3TTM RV Cone Plate (DVTRVCP) Rheometer (Toronto, ON, Canada), using the CPA-40z cone spindle (viscosity range of 1.7–32,700 cP) and the Rheocalc T® software (version 1.1.13). Measurements were performed at controlled temperature using a thermostatic water bath (MultiTemp III Thermostatic Circulator, Thermo Fisher Scientific, Waltham, MA, USA). Before viscosity measurements, equipment calibration was verified using Ametek Brookfield Fluid 500 Viscosity Standard (Middleborough, MA, USA) with a standardized viscosity of 489 cP at 25 °C. At each velocity, viscosity was registered after the spindle had enough time to perform five complete rotations. For fluids with Newtonian rheological behavior, viscosity was determined at the shear rate corresponding to the highest torque value (just below 100%), due to the equipment’s higher resolution and precision resulting in a lower measurement error. For non-Newtonian fluids, zero shear viscosity was estimated by the Y-intercept of the non-linear regressions based on the measurements performed at different shear rates at a constant temperature.
2.6. In Vitro Release of Model Drug
In vitro drug release studies, performed in horizontal Ussing Chambers (Harvard Apparatus, NaviCyte, Hugstetten, Germany), used phenytoin as a model drug. The methodology was adapted from Pires et al. [
13]. Synthetic membranes were used, with a pore size of 0.2 μm (hydrophilic polyethersulfone Supor
® membrane disc filters, Pall Life Sciences, MI, USA). The receiving chamber was filled with 1.8 mL of phosphate buffer (pH 7, 20 mM), plus albumin at 1%
w/
w, and 100 μL of this same solution was placed on the donor side of the membrane. The temperature was kept at 32 °C, the approximate temperature of the nasal cavity at the region of the middle turbinate [
14], with a heating water bath (Grant Instruments, Cambridge, England), and receiving chamber homogenization was achieved through magnetic steering (Micro Stirring Bars, 2 mm, VWR, United Kingdom). When the intended temperature was reached, the buffer on the donor chamber was replaced with 100 μL of the test formulations. Afterwards, samples of 100 μL were taken from the receiving chamber and replaced by the same volume of buffer plus albumin solution at 10, 20, 40, 60, 90, 120, 180, and 240 min.
For phenytoin quantification, samples were diluted 5-fold in a water-Transcutol mixture (3:1), followed by addition of perchloric acid at 10% (
v/
v). Formulation’s initial drug concentration was also quantified in a similar way, but the dilution was 500-fold. A previously developed and validated HPLC method [
15] was used, comprising specific apparatus and conditions: LC-2010A HT Liquid Chromatography system, coupled with a SPD-M20A diode-array detector, controlled by LabSolutions (version 5.52) software (Shimadzu, Kyoto, Japan); analyte separation was performed in a reversed-phase column (3 μm particle size, 55 × 4 mm) protected by a guard column (5 μm particle size, 4 × 4 mm, C18, LiChroCART
® Purospher
® STAR, Merck, Darmstadt, Germany), with isocratic elution at 1 mL/min at 30 °C. Mobile phase was a mixture of 36% (
v/
v) methanol and 64% (
v/
v) sodium phosphate buffer (10 mM, pH 3, with 0.25% triethylamine), filtered (Nylaflo membrane, 0.2 μm pore size, Pall, NY, USA) and degassed in a ultrasound bath. Sample injection volume was 20 μL, and phenytoin was detected at 215 nm, with a retention time of 10–11 min, within 20 min runs.
2.7. Cell Culture and Cytotoxicity Evaluation
Normal Human Dermal Fibroblasts (NHDF, adult donor cells, Ref. C-12302 from PromoCell) were cultured in RPMI 1640 medium supplemented with inactivated fetal bovine serum at 10%, 2 mM L-glutamine, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 1 mM sodium pyruvate, and 1% antibiotic (10,000 U/mL penicillin G, 100 mg/mL streptomycin) at 37 °C in a cell incubator with a humidified atmosphere of 5% carbon dioxide. The culture media was renewed every two or three days, and cells were subcultured as required.
To evaluate formulations’ cytotoxicity, 15,000 cells were seeded in 100 µL per well of 96-well culture plates the day preceding the experiment. Immediately before the experiment, the medium was replaced by 50 µL of complete fresh medium plus 50 µL of NE preconcentrates diluted in fresh medium at twice the concentration selected for treating the cells. Cells were then incubated for 30 min, after which the treatment medium was replaced by 200 µL of 10% (w/v) solution of resazurin prepared in Krebs Ringer Buffer (NaH2PO4.H2O 1.5 mM; Na2HPO4 0.83 mM, KCl 4.86 mM; NaCl 119.78 mM; MgCl2.6H2O 1.67 mM; NaHCO3 15 mM; anhydrous D-Glucose 10 mM; CaCl2.2H2O 1,2 mM) and plates incubated at 37 °C for the required time (until negative control wells had approximately a reference fluorescence level—10,000 RFU in our plate reader, as previously optimized). The fluorescence intensity of plate wells was measured using an excitation wavelength of 570 nm and emission wavelength of 590 nm in a microplate fluorophotometer (Spectramax GeminiTM EM, Molecular Devices, LLC, San Jose, CA, USA). After subtracting background fluorescence (wells with blank resazurin solution), cell viability data were expressed as a percentage of the negative control of non-treated cells. Each experiment was carried out in quadruplicate wells.
2.8. In Vivo Pharmacokinetic Study of Model Drugs through Intranasal or Subcutaneous Administration
Healthy adult CD-1 mice (8 to 11 weeks old, 39 to 42 g), originated from Faculty of Health Sciences’ certified animal facility were housed with controlled environmental conditions (12 h light/dark cycle, 20 ± 2 °C, 50 ± 5% relative humidity). Mice had free access to acidified tap water and standard rodent diet. All experimental procedures were carried out in conformity with the regulations of the European Directive 2010/63/EU and approved both by the Local Animal Ethics Committee and the competent national authority [Portuguese National Authority for Animal Health, Phytosanitation and Food Safety (Direção Geral de Alimentação e Veterinária)].
Mice were randomly divided into 2 groups, receiving either intranasal or subcutaneous administration (12 animals, 3 time points, 2 mice per time point). All animals were anesthetized with an intraperitoneal dose of pentobarbital (60 mg/kg) prior to formulation administration. All administrations were carried out with the mouse’s body lying on top of a heating pad (plus a DC Temperature Controller 40-90-8D, FHC, Bowdoin, ME, USA). The established administration volume was of 5 μL and 50 μL per 30 g of body weight, for intranasal and subcutaneous administrations, respectively. Afterwards the mice were left to recover in a supine position, in a temperature-controlled environment.
Euthanasia was conducted at specific time points (30, 240 and 720 min), after which mice blood and brain were collected. The blood was collected into K3 EDTA tubes (FL Medical, Italy), after which 300 μL were mixed with orthophosphoric acid 85% (v/v) in a 1:1 (v/v) blood/acid ratio. The brains were homogenized (Ika Ultra-Turrax® T25 Basic, Staufen, Germany) in a water-orthophosphoric acid [1:1 (v/v)] mixture (1 g of tissue per 4 mL of mixture), centrifuged (14,000 rpm, 4 °C, for 10 min, MIKRO 200R microcentrifuge, Hettich, Tuttlingen, Germany), and the supernatants were collected. Both acidified blood and brain homogenates were kept on ice and then stored at −20 °C.
Sample processing consisted of the addition of 20 μL of ketoprofen spiking solution (internal standard) to the sample (100 μL of acidified brain homogenate supernatant or 200 μL of acidified blood), followed by liquid–liquid extraction [addition of 1000 μL of diethyl ether, followed by vortexing for 30 s, and then by centrifugation for 5 min at 13,500 rpm at room temperature in a tabletop microcentrifuge (Gyrozen, Daejeon, Republic of Korea)]. Next the organic phase was transferred to a glass tube, and the aqueous phase was reextracted two more times, under the same conditions. The combined organic phases were then evaporated to dryness (gas stream, 45 °C) and reconstituted with 100 μL of mobile phase. The mobile phase was made of 36% (
v/
v) methanol and 64% (
v/
v) sodium acetate buffer (10 mM, pH 5, with 0.25% triethylamine). In each sample, phenytoin quantification was performed by HPLC, using a previously developed and validated method [
15], and aside from a change in mobile phase composition most chromatographic apparatus and analyte separation conditions were the same as described in
Section 2.6. for the drug release study. The only other parameter that was modified was the analytes’ detection wavelength, which remained 215 nm for phenytoin and fosphenytoin, but was changed to 280 nm for the internal standard.
2.9. Statistical Analysis
Data are represented by means of replicate measurements or of independent formulations ± standard deviation. Cell viability data were fitted by a nonlinear regression model [log(inhibitor) vs. normalized response—Variable slope] using GraphPad Prism version 9.5.1 to determine half-inhibitory concentrations (IC50).
4. Discussion
Several related low energy methods of NE production have been described [
18]. The most closely related to the process we used here with this novel NE is the reverse phase emulsification method, also named phase inversion emulsification [
19]. We claim this because we observed smaller and more homogenous droplet size by adding the aqueous phase in two steps (a very low amount first to force the formation of a water-in-oil emulsion, and then the rest to inverted it to oil-in-water) than in a single step. Since long ago, this strategy has been used in the traditional continental method of oral emulsion preparation to obtain finer droplet dispersions [
20]. This is also one of the reasons why we describe our system as a NE and not a microemulsion, since for microemulsions formation the order of excipients addition is not supposed have an effect.
Temperature is known to induce phase inversion of some emulsions with neutral surfactants (phase inversion temperature or PIT method [
21]), and refrigeration is known to increase emulsions’ physical stability. However, a particular composition that spontaneously forms smaller and more homogeneous droplets upon refrigeration without phase inversion has, as far as we know, not been previously described. The fact that after dilution (at least 10-fold) the small and homogeneous size is maintained at room temperature is likely due to the slower rearrangement of droplets at a greater distance.
It was also very surprising that the optimum mass proportion of oils to hydrophilic surfactant was between four and six to one. In fact, it is important to emphasize that the optimized preconcentrate has a composition of 83% of lipids or, more precisely, water-insoluble surfactants, and only 17% of a hydrophilic surfactant, which has no correspondence in the classification of lipid based formulations by Colin Pouton [
22]. The explanation of why the aqueous dispersion of this particular composition has this behavior and how refrigeration or the composition of the aqueous phase can modulate a droplet’s size and PDI is beyond the scope of our work. What has been shown, however, is how narrow the optimum design space seems to be.
Regarding the potential utility of the NE or of its preconcentrate, much remains to be explored in future works. One of the drugs that was demonstrated to solubilize at high strength in this vehicle was simvastatin, a lipophilic statin with low oral availability. This is a drug with pleiotropic effects and many potential applications beyond the control of dyslipidemia, like anti-cancer [
23], anti-fibrotic [
24], neuroprotective [
25,
26], and bone regeneration [
27] applications, among others. Some of these applications could benefit from an alternative liquid formulation for, for example, intranasal administration, which has the potential to enhance brain delivery [
28]. However, a limiting factor should be the relative cytotoxicity of the NE lipids Capryol 90 and Imwitor 948 compared to other lipids, as shown here. In fact, both Capryol 90 and Imwitor 948 are approved for cutaneous and oral administration and not for parental use. Therefore, the cutaneous and oral use of this NE, or of the preconcentrate as a self-nanoemulsifying drug delivery system, should not pose safety concerns since all excipients are well established and approved for these routes. Therefore, many possible applications could be envisioned in cosmetic or oral medicinal products. However, their safety for intranasal, ophthalmic, vaginal or parenteral delivery must still be established. For these routes, it is likely that there are limitations to the concentration/dose of the vehicle itself that can be safely used. Nevertheless, given the high content in Capryol 90:Imwitor 948, the studied NE might have the potential to provide a means to obtain a high concentration of lipophilic substances with a good solubility profile in this oil mixture. When compared to other oils, Capryol 90, in higher proportion in our formula, has shown to be the better solubilizer (among those tested) for very lipophilic drugs such as simvastatin (105 mg/mL [
29]), clofazimine (18 mg/mL, [
30]), luliconazole [~75 mg/mL [
31]), terconazole (116 mg/mL [
32]), tolvaptan (11 mg/g [
33]), and even novel drug candidates such as JIN-001 (41 mg/mL [
34]) and AC1497 (45 to 50 mg/mL [
35]), to mention just a few examples. Precipitation upon dilution is not expected to occur since no water-miscible co-solvents account for the initial drug solubilization, and droplet size does not reduce upon dilution and is shown to provide a slow release of phenytoin used as the model drug. Furthermore, the small size and optimal homogeneity are in favor of long-term physical stability for these compositions. As we have demonstrated, it can also promote the mucosal absorption of hydrophilic substances. This effect was similar to what was previously observed by us with a microemulsion of fosphenytoin [
36].