Phosal^® Curcumin-Loaded Nanoemulsions: Effect of Surfactant Concentration on Critical Physicochemical Properties

Abstract

Curcumin is a well-known and widely used substance of natural origin. It has also been found to be helpful in the treatment of liver diseases. Unfortunately, curcumin has very low bioavailability and a sensitivity to external agents. Improving these parameters is the subject of many studies. One way to overcome these problems may be to use Phosal^® Curcumin as a source of curcumin and encapsulate this dispersion into a nanoemulsion using different types and concentrations of surfactants and co-surfactants, thus manipulating the physicochemical parameters of the nanoemulsion. The present study aimed to develop curcumin-loaded nanoemulsions for intravenous administration and to investigate the effect of Kolliphor HS15 concentration on their critical quality attributes. Methods: Phosal^® Curcumin-loaded nanoemulsions with different concentrations of Kolliphor HS15 were prepared by high-pressure homogenization. The effect of Kolliphor HS15 on emulsion physicochemical properties such as mean droplet diameter (MDD), polydispersity index (PDI), zeta potential (ZP), osmolality (OSM), and pH, as well as encapsulation efficiency (EE) and retention rate (RR) of curcumin, were determined. Mid-term stability studies and short-term stress tests were conducted to evaluate the impact of Kolliphor HS15 on the critical quality attributes of the curcumin-loaded nanoemulsions stored under various conditions. Results: Five nanoemulsions with increasing Kolliphor HS15 concentrations were developed. Their MDD ranged from 85.2 ± 2.0 to 154.5 ± 5.1 nm, with a PDI from 0.18 ± 0.04 to 0.10 ± 0.01 and ZP from −15.6 ± 0.7 to −27.6 ± 3.4 mV. Depending on the concentration of Kolliphor HS15, the EE ranged from 58.42 ± 1.27 to 44.98 ± 0.97%. Conclusions: The studied parameters of the developed nanoemulsions meet the requirements for formulations for intravenous administration. Using the appropriate concentration of Kolliphor HS15 allows for a formulation that presents a protective effect against both curcumin and emulsion degradation.

1. Introduction

Curcumin (CUR) is a well-known natural compound isolated from turmeric (Latin: Curcuma longa, family Zingiberaceae). CUR has a long history of being used as a traditional remedy in Chinese and Ayurveda medicine [1]. Recent studies have demonstrated various therapeutic activities for CUR, such as anti-inflammatory, antioxidant, anticancer, immunomodulatory, antimicrobial, and neuroprotective properties [2,3]. CUR is considered a relatively safe compound, and the range of oral doses that is considered safe is very wide. No adverse effects have been demonstrated after oral administration in humans at doses as high as 1125, 1200, 2500, and even up to 8000 mg daily during several months of therapy [4,5,6]. Unfortunately, its bioavailability after oral administration is very low. Vareed et al. [7] showed that after a single oral dose of 12,000 mg of CUR, the free form was barely detectable in human plasma [7]. Other studies in rats showed that the absolute oral bioavailability of CUR powder was about 1% [8,9]. Moreover, CUR shows low stability in alkaline environments and at elevated temperatures. This poses an additional problem when developing a suitable drug formulation [2].

Two main steps have been taken to improve the bioavailability of CUR: modification of the excipient and the delivery system, which has increased the bioavailability by up to several tens of times [1]. One method to change the delivery system is to incorporate CUR into the nanoemulsion (NE) system. In vitro digestion studies have shown that incorporating CUR into NEs can significantly increase its bioavailability and bioaccessibility [10,11]. However, given the very low bioavailability of pure CUR, the improvement of CUR’s bioavailability through oral administration is not sufficiently satisfactory for a clinical therapeutic effect, and the problem of CUR absorption has not been substantially solved. Hu et al. [12] prepared a CUR injection system, and intravenous bioavailability parameters was compared with those of an oral CUR suspension. The concentration of CUR in the intravenous preparation was 20-fold lower, and after administration, the C_max and AUC were approximately 200-fold and 8-fold higher, respectively [12]. This allows us to conclude that the intravenous route of administration is a much more suitable and effective route for CUR.

Regarding modifying the excipients used, an effort was made to design several soluble CUR preparations, including liposomal systems, micellar systems, and complexes with cyclodextrins or phospholipids [8]. Phospholipids are an interesting approach to developing drug delivery systems as they can be used in the development of stable and safe drug dispersions. Despite phospholipid drug delivery systems’ many advantages, only 2–4% of commercially available drug products are based on this technology [13]. Allam et al. [3] designed an oral curcumin self-nano phospholipid dispersion based on the commercially available products Phosal^® 50 PG and Phosal^® 53 MCT (Lipoid, Ludwigshafen, Germany). They concluded that these combinations increase the oral bioavailability of CUR and may be new alternative carriers [13]. Given the above, it seems an interesting idea to use Phosal^® Curcumin (Phosal CUR), which is a liquid formulation consisting of about 7% CUR and about 50% purified sunflower phosphatidylcholine, dissolved in medium-chain triglycerides (MCTs). Phosphatidylcholine, due to its amphiphilic properties, disperses lipophilic curcumin. In addition, the formation of micelles can potentially protect the sensitive curcumin from degradation at elevated temperatures during the manufacturing or sterilization process and improve the bioavailability of CUR [13,14].

The wide-ranging properties of CUR have been the subject of many studies in which its therapeutic potential has been applied to the treatment of various conditions, such as lung diseases, neurodegenerative disorders, sepsis, and cancers of organs such as the lung, bone, cervix, breast, prostate, and liver [15,16,17,18]. Moreover, there are reports that by inhibiting oxidative stress, CUR can protect and treat liver disease and alter various cellular pathways [19]. Liver diseases such as liver injury, non-alcoholic steatohepatitis, non-alcoholic liver disease, liver fibrosis, and cirrhosis are very often caused or aggravated by oxidative stress accompanying liver damage caused by factors such as alcohol, drugs, viral infections, environmental pollutants, or dietary components. CUR is a leading compound in preventing and treating oxidation-related liver disease [20]. Farzaei et al. [19] have compiled a number of papers presenting experimental evidence that CUR exhibits preventive and therapeutic effects against oxidation-related liver disease through various cell signaling pathways [19]. Furthermore, the combination of CUR and phosphatidylcholine can not only alleviate the formulation problem but also provide many health benefits. Combining these two substances can lead to synergistic effects, such as preventing inflammation, oxidation, and liver disease [14]. The above information prompted us to develop a CUR delivery system suitable for intravenous administration, enabling potential use in several disease entities, including liver disease.

NEs are well-known, well-tolerable, safe, and biodegradable nanocarriers [2]. Since the 1960s, they have been successfully used in treatments as a source of non-protein energy in parenteral nutrition and as a carrier of poorly soluble lipophilic drugs [21]. The classification of NEs in terms of lipid droplet size is still unspecified, and one can encounter an acceptance range starting as high as 1000 nm. However, this limit is most commonly found to be set at 100–500 nm [22]. Commercially available NEs base their formulation on egg yolk lecithin as an emulsifier and have an average particle size between 200 and 300 nm [21]. It is worth mentioning that the NE preparation process is simple, and production is easy to scale up [23].

This study aimed to achieve a droplet size of less than 200 nm, and lowering this value was accomplished by using a co-emulsifier. One approved as safe by the FDA for intravenous use is (KOL) [24]. It is characterized by a high tolerance when administered intravenously, and it has already been incorporated into commercial products due to its strong solubilization properties in injectable formulations [25,26]. The formulation and development of NEs is a multi-step process. Therefore, based on the Quality by Design method, critical quality attributes (CQAs), which are the measurable characteristics that determine the performance of developed NEs, were defined [27]. The following CQAs were adopted: particle size expressed as the mean droplet diameter (MDD) and parameters such as polydispersity index (PDI), zeta potential (ZP), osmolality (OSM), encapsulation efficiency (EE), and pH.

The present study aimed to develop an intravenous NE containing Phosal CUR as a source of CUR and pure phosphatidylcholine, the combination of which, to the best of our knowledge, has not been tested at the present time. We also aimed to investigate the effect of KOL concentration on CQA, stability, and compatibility with infusion fluids. Considering the low bioavailability after oral administration and the high safety of CUR, as described above, the final concentration was set to about 0.5 mg/mL, allowing for the intravenous administration of a daily dose of up to 100 mg/day of CUR using 200.0 mL of NE.

2. Materials and Methods

2.1. Materials

Phosal CUR, soybean oil, Lipoid^® E80, and sodium oleate were kindly donated by Lipoid GmbH (Ludwigshafen, Germany). Water for injection, 0.9% NaCl, and a 5% glucose solution were purchased from B. Braun Melsungen AG (Melsungen, Germany). KOL and CUR were purchased from Sigma-Aldrich (Taufkirchen, Germany), and glycerol was purchased from POCH S.A. (Gliwice, Poland). All chemicals were analytical or high-performance liquid chromatographic grade.

2.2. Preparation of the CUR-Loaded NE

The five CUR-loaded NEs were prepared by high-pressure homogenization preceded by ultrasonic homogenization. A final concentration of 0.5 mg/mL of CUR was applied to the test preparations based on Phosal CUR as the CUR source. Phosal CUR is a liquid MCT-based formulation that contains 50% sunflower phosphatidylcholine and 7% CUR. The compositions of the formulations NE-1–NE-5, with increasing concentrations of KOL from 0 to 5%, are shown in

Table 1. Composition of CUR-loaded NEs.

Component (% w/w)	NE-1	NE-2	NE-3	NE-4	NE-5
Soybean oil	10.0	10.0	10.0	10.0	10.0
Phosal CUR	0.715	0.715	0.715	0.715	0.715
Lipoid® E80	1.2	1.2	1.2	1.2	1.2
Kolliphor HS15	-	0.25	1.0	2.5	5.0
Glycerol	2.25	2.25	2.25	2.25	2.25
Sodium oleate	0.05	0.05	0.05	0.05	0.05
Water	85.785	85.535	84.785	83.285	80.785

. The oil phase consisted of soybean oil and Phosal CUR, while the aqueous phase consisted of KOL, sodium oleate, glycerol, and water. Briefly, the phases were heated to a temperature of 60 °C on a magnetic stirrer at 600 rpm for 40 min. After dissolution, the oil phase was slowly dropped into the aqueous phase during continuous stirring on a magnetic stirrer. The resulting mixture was then transferred and homogenized using an ultrasonic homogenizer (Sonoplus HD 2070, Bandelin electronic GmbH & Co. KG, Berlin, Germany) at an amplitude of 50% (60 s on, 30 s off) for 12 min to obtain a coarse emulsion. The prepared emulsions were subjected to high-pressure homogenization under 1000 bar in 15 cycles (GEA PandaPLUS 2000, GEA Niro Soavi, Parma, Italy). The process parameters were based on methodologies found in scientific articles and adjusted during the preliminary research [28,29,30]. During this process, the formulations were cooled on ice. All NEs were stored at a temperature of 4 ± 1 °C without exposure to light. CUR-loaded NEs were subjected to heat sterilization and aseptic filtration. Thermal sterilization was carried out in a steam sterilizer for 20 min at 121 °C. For sterilization at room temperature, the NEs were filtered through sterile 0.22 μm cellulose acetate membrane filters with a diameter of 25 mm (Qpore^®, Heidelberg, Germany).

2.3. Physicochemical Characterization of NEs

2.3.1. Particle Size and Polydispersity Index Determination

The mean droplet diameter (MDD) and polydispersity index (PDI) of the NEs were determined using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK) based on the dynamic light scattering method. By varying the intensity of the laser light scattered by the particles and recorded by the detector, the intensity, volume, and number weighted distribution of the particles in the range from 0.3 nm to 10 µm and the PDI are determined. The instrument is equipped with a laser of 633 nm at a fixed scattering angle of 173°, and the temperature of the detection cell was kept constant at 25 °C during the measurements. Each sample was diluted 1:100 with water for injection, transferred to a polycarbonate cuvette, and placed in the detection cell. All measurements were performed in triplicate and the intensity-weighted particle size results are presented in this manuscript.

2.3.2. Zeta Potential Evaluation

The zeta potential (ZP) was determined at 25 °C using a Zetasizer Nano ZS (Malvern Instrument Ltd., Malvern, UK). Before measurement, the samples were diluted 1:100 with water for injection. The ZP determines the surface charge of the NE particles, and the laser Doppler electrophoresis (LDE) technique was used to determine it. Laser light is transmitted and scattered by charged particles moving at different velocities under the influence of an applied electric field. Based on the electrophoretic mobility of the micelles, the ZP value is determined using the Smoluchowski equation. All measurements were performed in triplicate.

2.3.3. pH Measurement

The pH of the obtained formulations was determined using a SevenCompact pH meter (Mettler Toledo, Columbus, OH, USA). Before the measurements, the pH meter was calibrated with standard buffer solutions (pH 4.00, 7.00, and 9.21). All measurements were performed in triplicate.

2.3.4. Osmolality Measurement

Osmolality was determined using an Osmometer 800 CLG (Tridentmed, Warsaw, Poland) using the freezing point method. The instrument was calibrated with distilled water. A volume of 100 µL of the tested NEs was placed in dedicated osmo-krio microtubes and inserted into the measuring head for osmolality determination. All measurements were performed in triplicate.

2.4. Quantitative Analysis of CUR in NEs

2.4.1. Spectrophotometric UV–Vis Measurements

UV–Vis spectrophotometry was applied to determine the concentration of CUR in the CUR-loaded NEs during the mid-term stability studies. The spectra were recorded at room temperature using a UV–Vis spectrophotometer (Lambda 20, PerkinElmer, Waltham, MA, USA). The following sample preparation was used: 100 µL of the CUR-loaded NEs was dissolved in 1 mL of dichloromethane, and the sample was filled up to 10 mL with methanol. Quartz cuvettes (1.0 cm) were used to record the spectra. The concentration of CUR was calculated based on a calibration curve ranging from 0.001 to 0.006 mg/mL, with absorbance determined at 421 nm. Standard stock solutions of pure CUR were prepared by dissolving the compound in methanol (1 mg/mL). The calibration curve was prepared using working solutions with concentrations ranging from 0.001 to 0.006 mg/mL.

2.4.2. High-Performance Liquid Chromatography Measurements

High-performance liquid chromatography (HPLC) was performed to evaluate the CUR concentration in the CUR-loaded NEs during short-term stress tests. The HPLC analysis was performed using an Agilent 1260 Infinity II LC System (Agilent Technologies, Santa Clara, CA, USA) equipped with a quaternary pump and degasser, a vial sampler set at 15 ± 2 °C, and a multi-column thermostat set at 35 ± 0.8 °C, coupled to a diode array detector (DAD). Elution was performed on the C-18 100 Å Luna^® column (150 × 4.6 mm ID, 5 µm; Phenomenex, Torrance, CA, USA), and isocratic solvent systems of 0.4% acetic acid (50%) and acetonitrile (50%) were used as the mobile phase at a flow rate of 1.0 mL/min and an injected sample volume of 10 µL. The determination of the CUR concentration in the NEs was performed as follows: a volume of 100 µL of the CUR-loaded NEs was dissolved in 1 mL of dichloromethane, and the sample was filled up to 10 mL with methanol. The obtained solution was injected into a chromatographic column. The time analysis of the chromatogram was set to 10 min, and the detection wavelength was adjusted to 420 nm. The concentration of CUR was calculated from the calibration curve ranging from 0.001 to 0.005 mg/mL. Standard stock solutions of pure CUR were prepared by dissolving the compound in methanol (1 mg/mL). A calibration curve was prepared with working solutions ranging from 0.001 to 0.005 mg/mL.

2.4.3. Encapsulation Efficiency (EE) and Retention Rate (RR) of CUR in NEs

The encapsulation efficiency (EE) of CUR was determined by direct measurements of its concentration in the CUR-loaded NEs. Test samples were prepared and analyzed using the HPLC method described in Section 2.4.2. The EE of CUR in the NEs was calculated using Equation (1):

$$EE\left[\%\right]={\displaystyle \frac{W_A}{W_T}}\times 100\%$$

(1)

where W_A is the amount of CUR determined in the NE and W_T is the total amount of CUR used for the preparation of the NE.

Following the methodology provided by Jin et al. [31], the retention rate (RR) was used to assess the chemical stability of CUR in the mid-term stability studies and short-term stress tests. The RR of CUR in the NE was calculated using Equation (2):

$$RR\left[\%\right]={\displaystyle \frac{C_t}{C_0}}\times 100\%$$

(2)

where C₀ is the CUR concentration at time zero, and C_t is the CUR concentration at subsequent sample collection times.

2.5. Stability Studies

To evaluate the physicochemical stability of the prepared NEs, the following tests were performed: mid-term stability studies at 4 ± 1 °C without exposure to light, short-term stability studies under stress conditions, and freeze–thaw tests.

2.5.1. Mid-Term Stability Studies

The effect of increasing the KOL concentration on the stability of the CUR-loaded NEs was studied under different storage conditions. The tested NEs were stored at a temperature of 4 ± 1 °C without exposure to light. An adequate aliquot of the NEs was withdrawn at fixed intervals (0, 3, 6, 14, 21, 28, and 130 days), and the chosen CQAs (MDD, PDI, ZP, pH, and osmolality) were analyzed. The RR of CUR in the NEs containing increasing concentrations of KOL was tested at temperatures of 4 ± 1 °C and 25 ± 1 °C, without light exposure. Samples for the mid-term stability studies were prepared and analyzed using the UV–Vis spectrophotometry method described in Section 2.4.1. An aliquot of 100 µL was taken at fixed intervals (0, 3, 11, and 60 days), and the RR was calculated as described in Section 2.5.3. All samples were prepared in triplicate, and the results are given as the mean ± standard deviation.

2.5.2. Short-Term Stress Tests

The CUR-loaded NEs were subjected to different stress conditions (heating, oxidation, alkalization, and acidification) to evaluate the stability of CUR and the NE itself. Samples for the short-term stress stability studies were prepared and analyzed using the HPLC method described in Section 2.4.2. To determine the stability of CUR at high temperatures, the CUR-loaded NEs were placed in a thermostatic chamber at 65 ± 1 °C without light exposure. For other stress factors, a 4 mL volume of the tested NEs was combined with equal volumes of oxidizing, alkalizing, and acidifying agents; these were 30% H₂O₂, 0.5 M NaOH, and 0.5 M HCl, respectively. The samples were stored at 25 ± 1 °C without light exposure for 7 days. For all tested samples at specific time intervals (0, 24, 48, and 168 h), pH and osmolality were measured, and the CUR concentration was determined. To investigate the RR of CUR, 100 µL (for reference and high-temperature effect samples) or 200 µL (for oxidative degradation, alkaline, and acidic environmental effect samples) of the CUR-loaded NEs was used to prepare working solutions. The samples exposed to alkaline and acidic environments were neutralized by adding 100 µL of 0.5 M HCl and 0.5 M NaOH solutions, respectively, before measurements. In addition, for all samples at the beginning and the end of the experiment (0 and 168 h), the MDD, ZP, and PDI were also determined. All samples were prepared in triplicate.

2.5.3. Freeze–Thaw Tests

To evaluate the effect of temperature changes on the physical stability of the NEs, the freeze–thaw test was performed using the Crystal1^TM multiple reactor setup (Technobis Crystallization Systems, Alkmaar, The Netherlands). The samples were mixed using magnetic bottom stirrers at a speed of 700 rpm and a temperature change rate of 0.5 °C/min. The temperature varied between −10 and 40 °C. The experiment was performed in three cycles, and after each cycle, the NE stability was determined by finding the differences in MDD, ZP, and PDI. The samples were then stored at 4 °C without light exposure, and the MDD, ZP, and PDI measurements were repeated after 28 days. All samples were prepared in triplicate.

2.6. Compatibility Studies

Compatibility was assessed by analyzing the physical parameters of the NEs after mixing with 0.9% NaCl or 5% glucose infusion fluids. Three dilutions of 1:1, 1:10, and 1:100 (v/v) were tested, and measurements were performed immediately after mixing and 3 h after mixing with the potential intravenous infusion. The measurement time was based on the recommended fastest intravenous supply of lipid emulsions—0.11 g of fat/kg BW/h [32,33] and the assumption of administering a CUR dose of 100 mg for a 70 kg patient. The given dose of CUR corresponds to 200 g of a 10% lipid emulsion and the shortest intravenous infusion, lasting 2 h and 36 min.

2.7. Statistical Analysis

The results are expressed as the mean value ± standard deviation (SD). The data were analyzed using Statistica 12 software (StatSoft, Krakow, Poland). One-way analysis of variance (ANOVA) was used to determine the statistical significance occurring among the samples. The a priori level of significance was p < 0.05.

3. Results

3.1. Characterization of NEs

The NEs developed in this study were formed using egg yolk lecithin with increasing concentrations of KOL, starting from 0 to 5%. As the co-surfactant concentration increased, the turbidity of the NE decreased. NE-1, without KOL, was characterized by a milky appearance with a light yellowish color due to the larger droplet size of the emulsion. As the concentration of KOL increased, the color became more intense, turning to orange, and the NEs became more transparent.

The methodology for the NE preparation was based on the results of preliminary studies involving test NEs without curcumin. This study tested the analogous procedure: phase mixing using a magnetic stirrer followed by ultrasonication, and then the application of high-pressure homogenization. The preliminary studies showed that ultrasonication was insufficient to produce the final emulsion. Therefore, it was decided to use this method to prepare a coarse emulsion. The MDD results obtained after ultrasonication were about 3 times higher than the final values. In addition, a second lipid droplet size fraction was detected, and the PDI for each emulsion was higher than 0.5, confirming insufficient homogeneity. The NEs’ MDD and PDI were also measured after five cycles of homogenization. The MDD values were 8% higher than the final values, but more importantly, the PDI was more than 30% higher than the final values. The results of the preliminary study are shown in Table S1.

Table 2. Characteristics of formulated CUR-loaded NEs.

	NE-1	NE-2	NE-3	NE-4	NE-5
CKOL [%]	-	0.25	1.0	2.5	5
MDD [nm]	154.5 ± 5.1	142.9 ± 2.6	114.2 ± 1.3	94.9 ± 5.1	85.2 ± 2.0
PDI	0.10 ± 0.01	0.10 ± 0.01	0.10 ± 0.01	0.12 ± 0.01	0.18 ± 0.04
ZP [mV]	−27.6 ± 3.4	−24.4 ± 1.4	−22.6 ± 0.7	−18.4 ± 1.7	−15.6 ± 0.7
EE [%]	44.98 ± 0.97	45.66 ± 0.53	50.36 ± 1.83	53.16 ± 1.44	58.42 ± 1.27
pH	7.47	7.78	7.75	7.62	7.51
OSM [mOsm/kg]	348	325	333	361	390

Results are presented as mean ± SD (n = 3). C_KOL—Kolliphor HS15 concentration; MDD—mean droplet diameter; PDI—polydispersity index; ZP—zeta potential; EE—entrapment efficiency; OSM—osmolality.

shows the CQAs of the tested NEs after the sterilization process, presented as the average value from two reproductive processes. Detailed data are shown in Table S2. Each prepared NE showed a satisfactory PDI below 0.18 and MDD in the 154.5 to 85.2 nm range. The particle size distribution of the developed NEs is shown in Figure S1. The absolute value of the external charge distributed at the oil droplet surface increased from −15.6 for NE-5 to −27.6 for NE-1. The osmolality values of the developed NEs ranged from 325 to 390 mOsm/kg, and the EE of NE-1–NE-5 ranged from 44.98% to 58.42%.

It is well known that CUR is susceptible to destabilization at temperatures above 70 °C [2,34], prohibiting thermal sterilization of its formulations. However, there are reports in scientific papers that NEs themselves can have a protective and stabilizing effect on unstable active substances of natural origin [35,36,37]. Therefore, we attempted to evaluate if the prepared NEs could act on CUR in this way. Two sterilizations were carried out on the NEs, thermal and aseptic filtration, to investigate the effect of the type of sterilization on the stability of the CUR-loaded emulsions. After thermal sterilization, visible emulsion delamination appeared in NE-3, NE-4, and NE-5. In NE-1 and NE-2, no emulsion structure changes were visible to the naked eye (Figure 1).

Each heat-sterilized NE was also tested to determine the MDD, PDI, and ZP. The MDD after heat sterilization reached values ranging from 194.0 to 969.3 nm, and the PDI ranged from 0.11 to 0.83. The ZP values reached values ranging from −20.3 to −27.6. A second lipid particle size fraction appeared in each developed NE. The limit pore size of the filter used for aseptic filtration was 22 μm, indicating that the NE lipid droplet diameter should not exceed this value to avoid qualitative and quantitative changes in the emulsion [2]. It can be concluded that aseptic filtration should be the sterilization method of choice for KOL-stabilized CUR-loaded NEs, which is more suitable than thermal sterilization.

3.2. Stability Studies

3.2.1. Mid-Term Stability Study

Mid-term stability studies were conducted to investigate the physical stability of the NEs at 4 ± 1 °C without light exposure for 130 days (Figure 2). Figure 2A shows the fluctuations of the ZP values at different time points during the study. Similarly, Figure 2B presents the changes in the MDD. Table S3 also shows the significant percentage change in the lipid droplet surface charge value after 130 days of storage.

The MDD of the developed NEs during storage at 4 ± 1 °C without light exposure after 130 days increased by 1.87%, 2.00%, 1.70%, 1.63%, and 0.00% for NE-1, NE-2, NE-3, NE-4, and NE-5, respectively (Table S3). Analyzing the results of the mid-term study, it was observed that the change in the MDD was not significant for NE-1 to NE-3 during the study period. For NE-4 and NE-5, a significant difference in the change in MDD values was noted only at days 7 and 21 and additionally at day 130 of the study for NE-4 (Figure 2B). The osmolality throughout the study period reached values ranging from 326.0 to 396.4 mOsm/kg, and the pH value decreased by an average of 2.0 pH points during storage for all the developed NEs (Table S4, Figure S2).

A UV–Vis spectrophotometric measurement method was used to determine the stability of CUR in the NEs during storage over 60 days at 4 ± 1 °C and 25 ± 1 °C without light exposure (Figure 3). It can be seen that the CUR was unstable and showed a very slight decrease in RR for each of the NEs tested under both conditions. The RR of CUR at 4 ± 1 °C dropped to a minimum of 98.2% for NE-3 and to a maximum of 92.4% for NE-1. These values were slightly lower for emulsions stored at 25 ± 1 °C; they dropped to 98.3% for NE-5 and 89.9% for NE-1 (Table S5).

3.2.2. Short-Term Stress Tests

A comparison of the effects of different stress conditions on the NEs was carried out during short-term stability studies. Each of the developed NEs was subjected to factors such as high temperature (65 ± 1 °C), oxidation (30% H₂O₂), acidic (0.5 M HCl), and alkaline (0.5 M NaOH) environments. The control samples were stored at 25 ± 1 °C in the absence of light for 7 days. The potential protective effect of the different KOL concentrations on CUR stability was determined by comparing the RR of CUR in each NE exposed to each of the stress factors; the results are presented in Figure 4.

A slight decrease in RR under acidic conditions was observed after the second day for the NE with the lowest KOL concentration (NE-2) and the NE without KOL (NE-1). For NE-3, NE-4, and NE-5, the RR values were 95.8 ± 1.1%, 95.8 ± 1.3%, and 93.5 ± 2.9%, respectively (Figure 4A). NE-3, NE-4, and NE-5 treated at 65 ± 1 °C showed RR values of 63.3 ± 1.2%, 69.3 ± 4.0% and 71.0 ± 0.9%, respectively, after 7 days. The RR values of CUR for the other two NEs were lower by more than 22% compared to NE-5 (Figure 4B). The most spectacular decrease in CUR stability was observed for the alkaline medium (Figure 4C). For each of the tested emulsions, the content of CUR decreased to almost 0. Nevertheless, it can be observed that the decrease was faster and more drastic for NE-1 and NE-2 immediately after combination with 0.5 M NaOH. Oxidative stress also destabilized the CUR in each emulsion, where the RR after 7 days was 77.2 ± 1.2%, 62.6 ± 0.9%, 74.5 ± 3.1%, 74.3 ± 1.4%, and 69.1 ± 1.0% for NE-1, NE-2, NE-3, NE-4, and NE-5, respectively (Figure 4D). The exact values of the observed RR changes are shown in Table S6.

The effect of stress factors on the stability of the NE system was also investigated. The factors used to monitor and determine the physical stability of the emulsion were the MDD, PDI, ZP, osmolality, and pH. The observed changes between the first and last measurements are shown in

Table 3. Physical stability of studied NEs under various stress conditions. The control sample was maintained at 25 ± 1 °C.

Sample	Environment	(t = 0 → t = 7 Day)
		MDD [nm]	PDI	ZP [mV]	Osmolality [mOsm/kg]	pH
NE-1	25 ± 1 °C	165.2 → 169.1	0.11 → 0.15	−25.8 → −28.2	314 → 314	6.9 → 6.3
	65 ± 1 °C	165.2 → 163.5	0.11 → 0.01	−25.8 → −32.4	316 → 313	6.9 → 6.3
	0.5 M NaOH	193.7 → 173.8	0.11 → 0.02	−5.0 → −32.1	605 → 503	12.7 → 12.6
	0.5 M HCl	327.0 →1791.0	0.44 → 1.00	−5.0 → −40.1	716 → 709	0.9 → 0.8
	30% H2O2	163.4 → 163.0	0.12 → 011	−26.7 → −24.6	-	4.9 → 4.2
NE-2	25 ± 1 °C	146.8 → 147.9	0.08 → 0.10	−24.9 → −25.7	330 → 332	7.2 → 6.2
	65 ± 1 °C	146.8 → 145.4	0.08 → 0.01	−24.9 → −31.8	330 → 336	7.2 → 6.5
	0.5 M NaOH	167.0 → 686.2	0.10 → 0.66	−6.3 → −32.0	599 → 528	12.8 → 12.6
	0.5 M HCl	172.4 → 435.2	0.13 → 0.49	−5.4 → −39.5	712 → 735	0.9 →0.8
	30% H2O2	145.0 → 149.0	0.10 → 0.10	−22.0 → −21.5	-	4.9 → 3.9
NE-3	25 ± 1 °C	113.3 → 112.3	0.11 → 0.09	−19.3 → 19.9	271 → 269	6.9 → 6.3
	65 ± 1 °C	113.3 → 112.4	0.11 → 0.02	−19.3 → −25.2	271 → 271	6.9 → 6.6
	0.5 M NaOH	111.2 → 247.6	0.09 → 0.40	−5.2 → −25.8	583 → 482	13.0 → 12.3
	0.5 M HCl	111.4 → 233.4	0.09 → 0.40	−4.5 → −27.9	677 → 694	0.9 → 0.8
	30% H2O2	113.4 → 111.9	0.09 → 0.11	−21.6 → −23.8	-	5.2 → 4.3
NE-4	25 ± 1 °C	98.7 → 97.8	0.11 → 0.11	−16.7 → −21.3	365 → 368	7.1 → 6.4
	65 ± 1 °C	98.7 → 97.2	0.11 → 0.11	−16.7 → −23.6	365 → 377	7.1 → 6.8
	0.5 M NaOH	97.6 → 268.6	0.09 → 0.35	−3.7 → −85.2	616 → 506	13.0 → 12.5
	0.5 M HCl	97.9 → 174.4	0.10 → 0.21	−6.1 → −27.6	726 → 740	0.8 → 0.9
	30% H2O2	98.0 → 97.4	0.11 → 0.10	−17.9 → −21.3	-	5.5 → 4.4
NE-5	25 ± 1 °C	84.0 → 83.4	0.13 → 0.15	−12.2 → −15.1	400 → 407	7.0 → 6.4
	65 ± 1 °C	84.0 → 88.0	0.13 → 0.12	−12.2 → −23.1	400 → 424	7.0 → 6.7
	0.5 M NaOH	83.2 → 822.0	0.12 → 0.75	−2.4 → −26.9	638 → 632	12.9 → 12.3
	0.5 M HCl	82.8 → 120.3	0.12 → 0.13	−5.7 → −23.4	747 → 769	0.8 → 1.0
	30% H2O2	82.9 → 82.3	013 → 0.14	−15.0 → −19.5	-	5.4 → 4.3

. The acidic and alkaline environments greatly affected the particle size and PDI but they varied with KOL concentration.

In the case of NE-1 (NE without KOL), immediately after adding 0.5 M HCl, the MDD value increased significantly to 327.0 nm with a PDI of 0.44. After 7 days, the MDD was over 1700 nm, becoming the main fraction of lipid droplets. The appearance of a second smaller fraction of particles with an average size of almost 68 nm was also registered. With this change, the PDI also increased to 1.0, indicating a destabilization of the emulsion. Under other stress conditions, no change in the stability of NE-1 was observed. On the test day, none of the stress factors caused significant changes in the MDD and PDI for emulsions NE-2, NE-3, NE-4, and NE-5. The situation changed dramatically after a period of 7 days. The MDD and PDI increased significantly in the samples containing KOL that were exposed to the alkaline environment after 7 days, and a second lipid particle size fraction appeared. NE-5, with the highest KOL concentration, showed the greatest increase in its MDD and PDI to over 820 nm and 0.75, respectively, with an accompanying second lipid particle fraction of a smaller size. The situation was reversed in the case of the acidic environment. As the KOL concentration increased, MDD and PDI decreased proportionally. As the average particle size decreased, there was still a second fraction of lipid particles, which for NE-3 and NE-4 reached a range of more than 4000 nm. Only NE-5 was a homogeneous emulsion, and the MDD after 7 days was 120.3 nm. Despite a gentle increase in the MDD of 37.5 nm since the start of the study, the measured size is still within the USP requirements [38]. The low PDI of 0.13 also confirms the homogeneity and stability of the NE-5 emulsion in the acidic environment after 7 days. Figure 5 graphically shows the differences in the MDD values for the NE without KOL (NE-1) and for NE-5 with the highest KOL concentration under acidic and alkaline conditions at t = 0 and after 7 days of testing. The numerical values are shown in Table S7.

Analyzing the surface charge of the droplets, significant differences can be seen for the samples treated with acidic and alkaline environments (Table 3). When combined with 0.5 M HCl and 0.5 M NaOH, there was a reduction in the absolute value of the ZP to single-digit numbers in each of the cases tested. After 7 days, the absolute value of the surface charge of the droplets increased significantly. This phenomenon was not observed when the emulsions were tested with the other stress factors. The pH values at the beginning and after the test did not change significantly for the NEs treated with an alkaline or acidic environment. No significant effect of different KOL concentrations was observed on the pH of emulsions subjected to acidic or alkaline environments, or oxidative stress. Unfortunately, the freezing point measurement method is unsuitable for determining the osmolality of samples subjected to oxidation.

3.2.3. Freeze–Thaw Tests

The freeze–thaw test was performed to evaluate the physical stability of the developed NEs. The MDD and PDI results are shown in Figure 6. The results of the freeze–thaw test showed no significant differences in the MDD and PDI for NE-3, both immediately after the test and after 28 days for each cycle (Figure 6).

3.3. Compatibility Studies

Compatibility studies with 0.9% NaCl and 5% glucose infusion fluids were conducted to test the hypothetical possibility of intravenous administration of the tested emulsions to the patient after reconstitution in the infusion fluids. The MDD, PDI, and ZP were measured at the time of combination and after 3 h, using different dilutions: 1-, 10-, and 100-fold. The results obtained by combining 5% glucose and 0.9% NaCl are shown in

Table 4. Results of compatibility tests with 5% glucose.

Sample	Ratio	MDD [nm]		PDI		ZP [mV]
		t = 0 h	t = 3 h	t = 0 h	t = 3 h	t = 0 h	t = 3 h
NE-1	1:1	152.5 ± 1.1	155.8 ± 2.0	0.09 ± 0.02	0.13 ± 0.02	−28.5 ± 1.4	−25.7 ± 0.4
	1:10	162.2 ± 2.4	189.7 * ± 2.5	0.13 ± 0.02	0.25 ± 0.01	−29.0 ± 2.0	−29.7 ± 1.3
	1:100	148.4 ± 2.5	280.9 ± 6.9	0.13 ± 0.02	0.24 ± 0.01	−25.1 ± 0.3	−27.6 ± 1.7
NE-2	1:1	143.3 ± 1.0	142.7 ± 2.1	0.09 ± 0.01	0.11 ± 0.00	−23.4 ± 0.1	−25.8 ± 0.3
	1:10	143.6 ± 0.7	146.6 ± 2.6	0.11 ± 0.03	0.12 ± 0.02	−23.9 ± 2.0	−22.6 ± 0.3
	1:100	148.6 * ± 8.2	-	0.19 ± 0.05	-	−24.3 ± 1.0	-
NE-3	1:1	114.4 ± 1.0	119.2 ± 2.4	0.11 ± 0.02	0.16 ± 0.03	−21.2 ± 0.6	−22.8 ± 1.7
	1:10	120.1 ± 0.6	122.5 * ± 2.6	0.15 ± 0.01	0.18 ± 0.03	−24.6 ± 0.4	−22.7 ± 0.4
	1:100	181.4 * ± 19.1	-	0.27 ± 0.02	-	−22.8 ± 1.0	-
NE-4	1:1	101.4 * ± 1.7	-	0.16 ± 0.01	-	−23.8 ± 0.7	-
	1:10	111.2 * ± 3.0	-	0.21 ± 0.01	-	−30.9 ± 1.6	-
	1:100	124.1 * ± 1.8	-	0.24 ± 0.01	-	−24.8 ± 0.9	-
NE-5	1:1	82.1 ± 1.2	88.9 * ± 0.5	0.14 ± 0.01	0.21 ± 0.01	−16.7 ± 0.6	−17.9 ± 0.8
	1:10	91.4 * ± 1.7	-	0.22 ± 0.01	-	−19.4 ± 0.5	-
	1:100	98.5 * ± 1.8	-	0.24 ± 0.00	-	−18.5 ± 0.2	-

*—The appearance of a second fraction of lipid droplets above 3200 nm. If the second fraction appeared after the first measurement, no second measurement was made after 3 h; MDD—mean droplet diameter; PDI—polydispersity index; ZP—zeta potential.

and

Table 5. Results of compatibility tests with 0.9% NaCl.

Sample	Ratio	MDD [nm]		PDI		ZP [mV]
		t = 0 h	t = 3 h	t = 0 h	t = 3 h	t = 0h	t = 3 h
NE-1	1:1	158.5 ± 1.2	169.0 ± 1.8	0.12 ± 0.01	0.14 ± 0.01	−29.1 ± 1.2	−23.9 ± 1.1
	1:10	163.7 * ± 3.5	-	0.17 ± 0.01	-	−28.6 ± 2.0	-
	1:100	179.2 * ± 2.9	-	0.19 ± 0.01	-	−32.3 ± 0.8	-
NE-2	1:1	142.5 ± 2.6	144.1 ± 1.5	0.10 ± 0.00	0.11 ± 0.01	−22.2 ± 0.7	−22.6 ± 1.2
	1:10	158.7 * ± 3.8	-	0.17 ± 0.00	-	−26.1 ± 0.8	-
	1:100	262.9 ± 4.7	186.5 * ± 4.1	0.18 ± 0.02	0.24 ± 0.01	−30.7 ± 0.4	−32.8 ± 0.7
NE-3	1:1	113.3 ± 2.4	112.5 ± 0.9	0.11 ± 0.01	0.11 ± 0.01	−17.6 ± 0.9	−18.8 ± 0.6
	1:10	115.2 ± 2.1	116.9 * ± 1.6	0.12 ± 0.02	0.16 ± 0.01	−15.5 ± 0.6	−26.8 ± 0.5
	1:100	143.6 * ± 6.3	-	0.24 ± 0.01	-	−18.6 ± 0.3	-
NE-4	1:1	96.7 ± 1.5	96.7 ± 0.6	0.10 ± 0.02	0.10 ± 0.01	−12.7 ± 1.3	−12.1 ± 0.4
	1:10	128.9 * ± 6.2	-	0.26 ± 0.03	-	−12.1 ± 0.4	-
	1:100	157.8 ± 3.5	152.0 * ± 1.2	0.24 ± 0.01	0.25 ± 0.01	−20.5 ± 2.9	−22.5 ± 0.5
NE-5	1:1	84.5 ± 1.3	80.6 ± 0.6	0.01 ± 0.02	0.12 ± 0.02	−10.8 ± 1.5	−9.0 ± 1.1
	1:10	105.7 * ± 0.2	-	0.27 ± 0.02	-	−7.8 ± 1.1	-
	1:100	149.1 ± 0.9	134.9 * ± 1.2	0.22 ± 0.02	0.27 ± 0.02	−11.6 ± 0.4	−24.8 ± 1.6

*—The appearance of a second fraction of lipid droplets above 3200 nm. If the second fraction appeared after the first measurement, no second measurement was made after 3 h. MDD—mean droplet diameter; PDI—polydispersity index; ZP—zeta potential.

, respectively. If emulsion destabilization with the appearance of a second droplet size fraction occurred after the first measurement, the second measurement after 3 h was not performed for this sample.

Analyzing the results of the emulsion compatibility test with 5% glucose, it is clear that for the emulsions with the highest concentrations of KOL, NE-4 and NE-5, destabilization occurred immediately or within the first 3 h of combination at each dilution tested (Table 4). This may indicate the incompatibility of KOL solutions at concentrations of 2.5% and higher with 5% glucose infusion fluid. NEs with KOL concentrations below 1% and without KOL showed compatibility with the 5% glucose solution at a 1:1 dilution, and NE-2 additionally did not show a second fraction of lipid droplets at a 1:10 dilution. The situation was different after emulsion reconstitution in 0.9% NaCl (Table 5). No effect of KOL concentration on emulsion stability after combination with 0.9% NaCl was observed. In each emulsion tested, compatibility was only maintained when combined with the infusion fluid in a 1:1 ratio.

4. Discussion

Due to its comprehensive and extended use in intravenous emulsions, soybean oil was chosen as the main ingredient in the oil phase. It is a reliable source of essential polyunsaturated fatty acids [39]. The combination of aqueous and oil phases requires the addition of surfactants, which stabilize the emulsion system by preventing the dispersed phase droplets from breaking in the dispersing phase. Lecithin, KOL, and sodium oleate were used as surfactants and co-surfactants to stabilize the NE system. Due to its high safety profile and use in commercial lipid emulsions, egg yolk lecithin, whose main chemical compound is phosphatidylcholine, was chosen as the main surfactant [40,41,42]. The amount of egg yolk lecithin was adjusted to 1.2% (w/v) according to FDA recommendations and the lecithin content of commercially available intravenous emulsions [24,39]. It is worth noting that Zhang et al. [43] indicated that the appropriate amount of egg yolk lecithin is 1.2% due to the formation of small lecithin particles, which significantly increases when used in excess [43]. KOL was chosen as a co-surfactant to reduce the MDD and improve the stability of the NE. In the preliminary studies, the emulsions were tested with selected co-surfactants registered for intravenous use by the FDA, i.e., KOL, Kolliphor^® ELP (purified Kolliphor^® EL), and Tween^® 80. The tests showed that emulsions with KOL had the highest formulation potential. This choice was also dictated by its high tolerance when administered intravenously and the fact that it has already been incorporated into commercial products due to its strong solubilization properties in injectable formulations [25,26]. Both emulsifiers were used at concentrations above their critical micellar concentration, which is 1.28 and 0.21 mg/mL for egg yolk lecithin and KOL, respectively [44,45]. Busmann et al. [46] noticed that increasing the mass share of KOL led to smaller MDD and narrower PDI distributions. A reflection of these observations can be seen in the measurements of MDD for NE-1–NE-5, where it is clear that the particle size decreased with increasing KOL concentration, ranging from no co-surfactant and a value of 154.5 ± 5.1 nm to 85.2 ± 2.0 nm for NE-5. When considering intravenous emulsion administration, it is essential to remember that the average capillary size is ~5–10 μm. Therefore, the MDD of an intravenous emulsion should be smaller than this size to prevent embolization of the blood vessels [47]. Moreover, according to the United States Pharmacopoeia (USP) requirements for injectable lipid emulsions and Method I of monograph <729>, the upper limit for droplet size is set at 500 nm, expressed as the MDD [38], which means that each of developed NE meets this requirement.

In contrast to Busmann et al.’s [46] observations, a value of 0.10 ± 0.01 for the PDI index was found for all the developed NEs with KOL concentrations up to 1% and increased slightly with increasing KOL concentration to 0.18 ± 0.04. The PDI index reflects the homogeneity and distribution of the oil droplets. The lower the PDI value, the more homogeneous the emulsion is. NEs with better stability will have a narrow and concentrated particle size distribution and the PDI value should be less than 0.2 [29]. This means that despite the slight increase in the PDI for NE-3–NE-5, it is still within the acceptable standard. Busmann et al. [46] also noted that using KOL in excessively high concentrations leads to cytotoxicity due to poorly water-soluble 12-hydroxystearic acid as a metabolic degradation product of KOL. They also established an upper limit of 21% for the KOL concentration and a minimum MDD of 50 nm, where exceeding these limits could induce a cytotoxic effect [46]. The results of these studies helped to establish a safe KOL concentration as being 5% or less.

It is well known that an increased surface charge causes greater repulsion of particles and thus stabilizes NEs. This parameter is used to determine the stability of colloidal systems, and it is assumed that a value above 30 mV (absolute) indicates a stable emulsion. However, the ZP value is limited, but analyzing the change in this value and the changes in the system can provide important information [48]. Moreover, adding a small amount of sodium oleate (0.05% (w/w)) significantly increased the absolute ZP value, without hemolytic activity [43,49]. Studying the ZP, it was found that the surfactant-coated particles were negatively charged, and the ZP of the NE without KOL was −27.6 ± 3.4 mV. As the co-surfactant concentration increased, the ZP value decreased significantly to −15.6 ± 0.7 mV. A similar phenomenon was observed by Chuacharoen and Sabliov, where the absolute ZP value decreased when a non-ionic co-surfactant was added to the lecithin-based nanoformulation [50].

The osmolality of the developed NE was controlled with glycerol so that its value was close to the physiological osmolality (297.5 mOsm/kg), bearing in mind that a hypertonic solution with a higher osmolality can cause significant pain and red blood cell crenation. Similarly, a hypotonic solution with an osmolality lower than 150 mOsm/kg can cause hemolysis and pain at the injection site [51]. Glycerol is recognized as a safe component since it is naturally present in the blood serum. It can be found in all commercially available intravenous lipid emulsions used for parenteral nutrition, as well as lipid emulsion-based propofol and etomidate formulations. The osmolality values of the developed NEs ranged from 325 to 390 mOsm/kg, suggesting good tolerability after intravenous administration. The EE of NE-1–NE-5 ranged from 44.98% to 58.42%, and the entrapment capacity of CUR increased with increasing KOL concentration and thus with decreasing MDD, as also reported by Chuacharoen et al. [34]. The EE of 44.98% to 58.42% is a thought-provoking level of encapsulation. Phosal CUR is a high-density, high-viscosity dispersion, which posed quite a challenge during the NE preparation process, both when measuring the mass and volume. Heating the product made it easier to measure it accurately, but due to the presence of thermolabile CUR, the temperatures could not be too high. The nature of the dispersion also raise questions about the guarantee of an even distribution of CUR in the preparation. These factors could have potentially affected the EE levels described.

Analyzing the results of the mid-term stability studies, it was observed that each NE fulfilled the USP standards for an injectable lipid emulsion during the duration of storage [38] in terms of the MDD. Despite some percentage changes occurring in the PDI coefficient during the test, it remained at low values, thus indicating adequate dispersion and homogeneity of each developed NE (Table S4). The ZP is also an important parameter for determining the surface charge of oil droplets. As its absolute value decreases, the risk of emulsion destabilization increases, resulting in coalescence and flocculation [43]. The developed NEs showed significant variation in the ZP values throughout the storage period, irrespective of KOL concentration. Nevertheless, the absolute value of the ZP decreased with increasing KOL concentration (Figure 2A), which may suggest the susceptibility of these NEs to destabilization.

Interestingly, a significant increase in the ZP was noted on day 130 of the study for NE-3 and NE-5 (Figure 2A). With the increasing duration of the study, the pH value of the developed NEs also decreased so that by day 130, it reached a value in the range of 5.8–5.2. It was noted that once the aqueous KOL solutions’ clouding point is exceeded, measured at 75–80 °C, the KOL becomes more dehydrated and tends to leave the interfacial layer and form micelles. This process results in a proportionally higher sodium oleate content on the surface of the oil droplet and can lower the ZP value [44]. Similar processes may occur during long-term NE storage with an accompanying pH decrease, which may explain the decrease in the ZP for NE-3 and NE-5 after 130 days of storage.

The intravenous emulsion should be euhydric to avoid or minimize local damage to blood vessels and cells. Local tolerance after intravenous administration is multifactorial, so no specific pH limits exist. Some authors accept, for small-volume intravenous injections (<100 mL), a range of values from 3.0 to 11.4, with the recommended values to avoid the risk of tissue damage being 5.5–8.5 [51]. For the intravenous administration of large volumes (>100 mL), different recommendations exist depending on the site of administration. Emulsions with pH values outside the 5–9 range can be administered into the vena cava, subclavian vein, and proximal axillary vein. Fluids whose pH does not exceed the range of 5–9 can be administered into the cephalic and basilar veins on the arms [52]. It is worth noting that the initial pH value is slightly higher for emulsions containing KOL in their composition. A more significant drop in pH was also observed for them. The obtained osmolality results showed that it did not change during storage under different conditions, regardless of the amount of KOL used to prepare the NE. Based on the above data, it can be concluded that the prepared NEs meet the requirements for intravenous fluids in terms of the MDD, PDI, ZP, pH, and osmolality throughout the storage period.

From the results of the tests of CUR stability in NEs during storage, a slightly lower protective effect for CUR was observed for the emulsions with a lower KOL concentration or without KOL. Nguyen et al. [53] showed a significant improvement in the solubility of CUR with KOL, which confirmed the EE% results obtained, where the value increased with increasing KOL concentration (Table 2). The results achieved during the mid-term stability study also suggest that the presence of KOL in the NE as a co-surfactant may increase the stability of the incorporated CUR.

By observing the stability of CUR under stress conditions, it was noted that in an acidic environment, CUR appeared to be relatively stable. The overall stability of CUR in the acidic media was due to the structure of the compound. The conjugated structure of the diene might contribute to the high stability under conditions of 0.5 M HCl [31]. A more significant decrease in the RR of CUR was observed at constant high temperatures. However, the protective effect of NEs with higher KOL concentrations on CUR stability was visible. As the temperature increases, the hydrolysis of the phospholipids used as emulsifiers of the emulsion also increases. This may affect the increased permeability of the micelles in which CUR is encapsulated and, as a result, may lead to leakage of CUR into the dispersing phase and accelerate CUR degradation [31,54]. The use of KOL as a co-emulsifier may have a stabilizing effect on the structure of the micelles and thus slow the escape of CUR from the micelles’ interior. Apart from a decrease in the CUR content after 7 days under alkaline conditions, there was also a significant change in the appearance of the NEs after adding 0.5 M NaOH. They changed color from yellow to an intense orange, and after 24 h, the consistency became gelatinous, which became liquid again after shaking. The jelly-like consistency became stronger and more difficult to reverse day by day. During the test, the intense orange color faded drastically. The destruction of the conjugated dienes in the structure of CUR is the reason for its rapid degradation in an alkaline environment, which starts above pH 7 [34]. Jin et al. [31] also noted the rapid disappearance of the color of the CUR solution until it was completely lost after 40 h, which was directly related to CUR degradation [31]. The dark orange color obtained after adding 0.5 M NaOH may be due to the Phosal CUR used to prepare the NEs, other excipients, and their interactions. However, the fading of this color during the degradation process of CUR remains unquestioned. Oxidative stress also destabilized the CUR in every emulsion tested; in this case, there was no effect of KOL concentration on CUR stability. The protective effect of KOL on CUR stability was most clearly observed under thermal treatment and in an acidic environment. Even though CUR degraded intensely and rapidly in an alkaline medium, the protective properties of KOL could also be observed, proportional to the increase in its concentration.

Observation of the effect of stress factors on the stability of the NEs allowed us to conclude that only acidic and alkaline environments affected their stability. In summary, regarding the effects of alkaline and acidic environments on the MDD and PDI, the emulsion without KOL (NE-1) appeared stable only in alkaline environments. In contrast, the emulsion with the highest KOL concentration (NE-5) remained stable only in acidic environments (Figure 4). The other emulsions with intermediate KOL concentrations (NE-2, NE-3, and NE-4) proved to be unstable under both conditions (Table 3). When analyzing the other two stress factors, high temperature and oxidation, no destabilizing effect on the MDD and PDI of the emulsions was observed. Many researchers have observed a relationship between pH, the surfactant used, and the droplet surface charge in emulsions [34,55,56,57]. It has been observed that lecithin-based emulsions show poor stability in acidic environments [55,56]. Rao and McClements [55] explained that the increase in the MDD and the aggregation process of emulsions at low pHs occurs through reduced electrostatic repulsion due to the preferential adsorption of hydrogen ions on the droplet surface [55]. A study by Yang et al. [57] showed that droplet aggregation in acidic conditions can be inhibited by steric repulsion. This occurs when a co-emulsifier with large hydrophilic head groups (e.g., KOL) is used, which will cause a net charge on the droplets at very low pH values [57]. The use of high temperatures also has a significant effect on increasing the absolute ZP value. In each of the NEs tested, the thermal treatment increased the external charge of the droplet, increasing the stability of each emulsion tested. In their study, Chuah et al. [58] showed that the strength of electrostatic repulsion increases when lecithin and co-surfactant are used together, and the effect is stronger as their concentration increases [58]. This may be another argument for the simultaneous action of KOL in stabilizing not only CUR but also the emulsion itself.

For NEs treated at 65 ± 1 °C, it was observed that the pH decrease was slower compared to the control (Table 3). For NE-3, NE-4, and NE-5, the difference in pH was lower than for NE-1 and NE-2, which may again confirm the protective effect of KOL on CUR and the emulsion itself against high temperatures. Stress factors, such as thermal treatment and acidic environments, affected the osmolality slightly, up to a maximum of 3.2% from the initial values, for each of the emulsions tested. Analyzing the osmolality changing under the influence of the alkaline environment after 7 days, a decrease in the range of 11.9–17.9% was observed for NE-1, NE-2, NE-3, and NE-4. A noticeable stabilization of osmolality was observed for the emulsion with the highest KOL concentration (NE-5), where it decreased by 5.7%compared to the initial values.

The results of the freeze–thaw tests showed that each emulsion was stable in terms of indicators such as the MDD, PDI, and ZP over the temperature cycles tested. No effect of the different KOL concentrations was observed on the emulsion stability during cyclic freezing and thawing.

Based on the compatibility studies of the developed NEs mixed with infusion fluids, each can be reconstituted in 0.9% NaCl at a dilution of 1:1 in an infusion lasting a maximum of 3 h. Choosing a 5% glucose infusion fluid as the medium to reconstitute the NE is only possible for NEs without KOL or with KOL concentrations below 1%, again at a 1:1 dilution.

5. Conclusions

In this study, we developed and characterized CUR-loaded NEs for intravenous administration with the use of Phosal CUR as the source of CUR. Our results also proved the effect of the co-surfactant concentration on the stability maintenance and selected CQAs of the NEs. The EE of CUR increased with increasing KOL concentration, which was inversely proportional to the MDD of the emulsion. However, in NEs with KOL concentrations above 1%, the homogeneity of the NE decreased slightly, as did the surface charge of the droplet. Aseptic filtration appeared to be a suitable sterilization technique for the tested emulsions containing KOL and CUR. The selected co-surfactant had a protective effect on the CUR in the NEs when subjected to stress conditions. The higher the KOL concentration, the slower the rate of CUR degradation. In conclusion, the use of appropriate amounts of co-surfactants may provide beneficial protection for entrapped CUR by maintaining the physicochemical stability of the emulsion and CUR itself. Moreover, the above data also allowed us to conclude that the measured parameters such as the pH, osmolality, ZP, PDI, and MDD of each prepared NE indicate that they meet the requirements for injection administration.