Introduction

In 2020, 1.93 million new cases of colorectal cancer (CRC) were globally diagnosed, and it is estimated that by 2040, 3.2 million new cases will have been reported [1]. Most CRC develop from a sporadic and benign abnormal proliferation of colon and rectum cells called polyps, which increase in incidence with age and may progress to invasive and metastatic forms [2, 3]. The treatment is based on surgery and radiotherapy; chemotherapy plays its role in advanced cases, using drug substances such as fluoropyrimidine, oxaliplatin, irinotecan, and capecitabine. However, chemotherapy may have unpleasant consequences such as systemic toxicity, unsatisfying response rate and acquired resistance. Targeted therapy may address these issues, but drawbacks are still present, such as the cost–benefit balance, adverse events, and variable efficacy among patients [4].

Hence, new forms of treatments and drug substances have become the theme of numerous research lines. As the costs of development and registering new drugs are exorbitant, drug repositioning emerges as an alternative, which consists of identifying new uses for approved or under-development molecules [5]. Niclosamide is a drug used for the treatment of tapeworm infections and its mechanism of action involves the uncoupling of oxidative phosphorylation in the parasite’s mitochondria [6]. This drug substance has shown promising anticancer activity by inhibiting oncogenic signaling pathways—such as Notch, mTOR, NF-kB, STAT-3, and Wnt/β-catenin—which, when unregulated, contribute to emergence and progression of cancers, justifying the claim for drug repositioning [7].

However, BCS class II niclosamide presents low water solubility and dissolution rate, which compromises its oral absorption and therapeutic action [5, 8]. Pharmaceutical strategies have been proposed to overcome these challenges. Examples include amorphous solid dispersions [9, 10], nanocrystals [11, 12], matrix nanostructured systems [13, 14], co-crystals [15, 16], and inclusion complexes [17, 18]. Nanoemulsion is one of the most promising pharmaceutical strategies among the nanostructured systems. The incorporation of a drug substance in nanostructured liquid lipid particles allows its administration already dissolved in the liquid lipid, which overcomes the dissolution step for oral absorption of BCS class II molecules. Moreover, the nanoscale enables optimization of tumor penetration of the drug. Nonetheless, challenges include the stability of a system with at least two immiscible components, which is constantly subjected to phase separation mainly due to (1) coalescence, when particles fuse to each other to reduce the interfacial area, or (2) Ostwald ripening, when larger particles grow due to mass transferred from the smaller particles [19].

Therefore, in this study, we aimed at the development, physicochemical characterization, and in vitro anticancer activity of a niclosamide nanoemulsion, with HCT-116 as the cell model.

Materials

Niclosamide (purity > 99%, Cayman Chemical, USA) was used as the drug. A variety of medium-chain liquid lipids were used: tryglicerides from capric/caprilic acids (Miglyol® 812, Captex® 300, and Captex® 355), propylene glycol dicaprylate/dicaprate (Miglyol® 840), glyceryl tricaprylate (Captex® 8000), mono, and diglycerides of caprilic acid (Capmul® MCM C8). Other lipids include a long-chain glyceryl trioleate (Captex® GTO) and oleic acid (Croda do Brasil Ltda). As oils, olive (Sigma Aldrich, USA), mineral (Volp Ind. Comércio Ltda.), corn, cotton, soybean, sesame, and safflower (Croda do Brasil Ltda.) were tested. Polyoxyl 40 hydrogenated castor oil (Kolliphor® RH 40), poloxamer 188 (Kolliphor® P188), polysorbate 80 (Tween™ 80) (Sigma Aldrich, USA), lauroyl polyoxyl-32 glycerides (Gellucire® 44/14), phosphatidylcholine (lipoid S100), and caprylocaproyl polyoxyl-8 glicerides (Labrasol®) (Gattefossé, France) were the surfactants. We are particularly thankful to Abitec Corporation (USA), IOI OleoChemical (Germany), Volp Ind. Comércio Ltda. (Brazil), Croda do Brasil Ltda. (Brazil), BASF (Germany), Lipoid (Germany), and Gattefossé (France), which kindly donated samples for this research. Other materials were: purified water, absolute ethanol, dimethyl sulfoxide (DMSO); magnetic stirrer bars, vials; polystyrene cuvette, glass flasks, cover glasses, and an Amicon® Ultra-0.5 10 kDa centrifugal filter.

Method

Niclosamide differential scanning calorimetry (DSC) and thermogravimetric (TG) analysis

Niclosamide DSC thermal behavior was characterized in a DSC-60 differential scanning calorimeter (Shimadzu, Japan) in a closed Al crucible. The sample mass was ~ 2 mg, and the scanning was set for 10 °C/min heating rate and 25–350 °C temperature range, under a dynamic nitrogen atmosphere (50 mL/min). TG analysis was carried out in a TGA-60 thermobalance (Shimadzu, Japan) in a platinum crucible, with sample mass of ~ 2.8 mg, under an N2 cycle atmosphere (100 mL/min), set for 10 °C/min heating rate and 25–600 °C temperature range.

Liquid lipid and surfactant selection

To select the liquid lipid for the nanoemulsion, Crystal16 (Crystal Pharmatech Inc., USA) equipment was used [20]. The objective was not only to select the liquid lipid for the nanoemulsion development, but also verify whether medium-chain lipids provide a better solubility medium for niclosamide than long-chain lipids. Two main groups of liquid lipids were tested: medium-chain (Miglyol® 812, Miglyol® 840, Captex® 300, Captex® 355, Captex® 8000, Capmul® MCM C8) and long-chain (Captex® GTO, oleic acid, and the olive, mineral, corn, cotton, soybean, sesame, and safflower oils). For this experiment, 1 g of each liquid lipid was weighed and added into 2-mL vials. Then, 10 mg of niclosamide was added, along with magnetic stir bars. Samples were submitted to heating from 25° to 80 °C, kept at 80 °C for 1 h, then cooled to 25 °C. The equipment was set for 0.5 °C/min heating/cooling rate, and 700 rpm stirring speed. The transmittance (%) of the sample was monitored throughout the test and indicated the solubilization capacity of the lipid.

The chosen lipid was used to prepare blank nanoemulsions for surfactant selection. Four types of surfactant were tested: Kolliphor® P188, Kolliphor® RH 40, Tween™ 80, and Gellucire® 44/14. The liquid lipid and purified water were fixed at 5% and 94% (w/w), respectively, and the surfactant was employed at 1% (w/w).

Preparation of the nanoemulsions

Formulations were prepared according to the following protocol: the components were weighed and transferred to beakers, one for the aqueous phase (containing surfactant) and the other for the lipidic phase (liquid lipid and drug). The system was heated to 80 °C and kept at this temperature under magnetic stirring (250 rpm); once the components were solubilized, the aqueous phase was added to the oil phase. The formulation was then subjected to premixing using Ultraturrax® at 13,000 rpm for 5 min, submitted to high-pressure homogenization (NanoDeBEE, BEE International, Inc., USA), set for 10,000 psi and 5 cycles/min, stored in glass flasks and kept under refrigeration at 4 °C and under room temperature.

Niclosamide nanoemulsion optimization

Following the preliminary tests for selection of nanoemulsion components, design of experiments (DoE) was used to optimize the formulation. Experimental design by surface response was obtained using MiniTab® 20 software. Two independent variables were considered: the percentages of liquid lipid and surfactant (%, w/w) (Table 1), including five central points, totaling 13 formulations. The objective was to verify the influence of these two factors on the particle size (hydrodynamic diameter). Statistical analysis was performed using MiniTab® 20 software (Minitab®, USA).

Table 1 DoE for nanoemulsion optimization. Niclosamide proportion (% w/w) was fixed, considering 10 mg of niclosamide/1 g of liquid lipid, for batches of 50 g
Full size table

Particle size, zeta potential, particle concentration, and stability study

To assess particle size and zeta potential, Zetasizer Nano ZS90® equipment (Malvern Pananalytical, UK) was used. Purified water was the solvent, and samples were diluted using 10 μL of the formulation to 2000 μL of solvent. Mean particle size (hydrodynamic diameter, HD) and polydispersity index (PDI) were determined by dynamic light scattering (DLS), at 25 °C and 90° angle. Zeta potential (ZP) was determined by electrophoretic mobility, according to Henry’s Eq. (1):

$$U_E=\frac{2\varepsilon\mathrm z\;\mathrm f\;\left(\mathrm ka\right)}{3\eta}$$
(1)

where UE = electrophoretic mobility, z = zeta potential, ε= dielectric constant, η= viscosity, and f(κa) = Henry’s function. For ZP determination, the field strength was 20 V/cm.

Particle concentration was measured by multiangle-DLS (MADLS) in Zetasizer Ultra Advanced (Malvern Pananalytical, UK) equipment, using a He–Ne laser at 633 nm wavelength and maximum power of 10 mW. The readings were performed in polystyrene disposable cuvettes at 25 °C, using different sample volumes (10–50 μL) diluted in purified water. Each point corresponds to the average total particle concentration (n = 12). The instrument settings were optimized automatically by ZS XPLORER software (Malvern Panalytical, UK).

For the stability study, formulations were stored in a climatic chamber (ClimaCell Eco-Line, Germany), set for 30 °C/75% RH, and also stored under refrigeration at 4 °C. At the end of each period, particle size, ZP, and PDI were determined using a Zetasizer Nano ZS90® (Malvern Instruments, USA), as previously described.

Encapsulation efficiency and drug loading

For this test, niclosamide was quantified by UV–vis spectrophotometry [21], using Evolution Series 201® (ThermoFisher, USA) equipment. Linearity was obtained at 331 nm wavelength in the range 3–17 μg/mL (y = 0.051x – 0.038, r2 = 0.9992), using absolute ethanol as medium.

For encapsulation efficiency (EE%), the mass of the free drug in the supernatant was estimated after centrifugation/filtration. For this, 0.5 mL of the nanoemulsion was transferred to an Amicon® Ultra-0.5 10 kDa centrifugal filter and submitted to mild centrifugation (centrifuge 5424 R, Eppendorf, Germany), set for 5000 g for 30 min. The filtrate was withdrawn, diluted in absolute ethanol and the absorbance was compared with niclosamide standard solution. EE% and drug loading (DL%) were then estimated according to Eqs. (2) and (3) [22], respectively:

$$EE\%= \frac{TD-FD}{TD}*100$$
(2)
$$DL\%= \frac{TD-FD}{TF}*100$$
(3)

where TD corresponds to the total amount of drug taken for formulation, FD corresponds to the amount of free drug in the supernatant, and TF to the total weight of the nanoemulsion, respectively.

Toxicity on Galleria mellonella larvae

Healthy Galleria mellonella larvae (2 to 2.5 cm in length and 150 to 200 mg in body mass) were chosen to carry out the toxicity study [23]. For this purpose, four samples were considered: niclosamide nanoemulsion (Nano-NCL, ~ 200 nm), placebo formulation (Blank, ~ 200 nm), free drug in DMSO (NCL-DMSO), and 5% DMSO in phosphate buffer solution (PBS). Then, 10 μL of each sample was injected, using Hamilton® syringes. A control group of larvae received PBS (16–20 larvae/group). Larvae were incubated at 37 °C and observed every 24 h for 5 days, to obtain survival and morbidity curves.

Formulation effects on HCT-116 cell viability

To assess the influence of niclosamide incorporation into the selected nanoemulsion on its cytotoxicity, HCT-116 cells (human colorectal carcinoma, American Type Culture Collection, Manassas, VA) were used as model. They were maintained in Dulbecco’s Modified Eagle Medium (DMEM) – F12™ medium (Gibco, Carlsbad, CA) with 10% fetal bovine serum, 100 U/mL penicillin and 100 U/mL streptomycin at 37 °C, and 5% CO2. For the experiments, they were cultured in 96-well plates containing 3 × 104 cells/well and treated with samples diluted in culture medium.

To verify the influence of nanoemulsion incubation in the culture medium on the particle size, a 5 mg/mL Nano-NCL dispersion in DMEM – F12™ culture media was prepared, added to a 12-well culture plate, and incubated at 37 °C and 5% CO2 for 24 and 48 h. The samples were collected to verify particle size as previously described and to assess the presence of crystals or precipitates by optical microscopy.

To evaluate the effect of treatment on HCT-116 cellular viability, samples containing 1.53 μmol/mL of niclosamide were used: free drug in DMSO (DMSO-NCL), nano formulation (Nano-NCL, 200 nm), unloaded formulation (Blank, 200 nm), and a niclosamide coarse emulsion (Coarse-NCL, particle size > 1 μm, PDI > 0.6) with the same composition as Nano-NCL. The preparation of this sample involved the same steps as the preparation of Nano-NCL, except that after mixing the aqueous and lipidic phases, the system was only subjected to premixing using Ultraturrax® at 13,000 rpm for 5 min.

Serial dilutions of the samples were performed in culture medium (10 μL/1000 μL) to obtain formulation concentrations of 5 to 0.16 mg/mL in the medium, which is equivalent to 7.5 to 0.23 μM of niclosamide. They were added to the 96-well plates with cells, and the plates were incubated for 24 and 48 h. Three hours before the end of the incubation period, the medium was replaced with culture medium containing 0.5 mg/mL of 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich), and the plates were incubated for 3 h. The medium was subsequently removed, and after drying, DMSO was added. The reading was performed on a plate spectrophotometer at 595 nm wavelength. The negative control was cells treated with DMSO, whereas the positive control was doxorubicin (Sigma-Aldrich).

Data were processed using Microsoft Excel software, using the negative control (untreated samples) as 100% viability. Results were then analyzed by a four-parameter nonlinear regression mathematical model using the GraphPad Prism 7 program (GraphPad Software, Inc., San Diego, CA, USA) and graphs were created using Origin 2019.

Formulation effects on the viability of 3D cell culture models

For this test, a set of in vitro experiments was carried out using spheroids formed by HCT-116 cells (American Type Culture Collection, Manassas, VA). The liquid overlay technique was used to obtain the spheroids in cell culture, which consists of not allowing the cell to adhere to the surface of the container [24]. For this, 200 μL of culture medium were placed over 50 μL of 1% agarose in 96-well U-bottomed culture plates containing 15,000 cells. The plate was centrifuged at 3000 rpm for 5 min immediately after plating and incubated for 48 h for spheroid formation.

Samples containing 1.53 μmol/mL of niclosamide were used: free drug in DMSO (DMSO-NCL), nano formulation (Nano-NCL, ~ 200 nm), placebo formulation (Blank, ~ 200 nm), and a niclosamide coarse emulsion (Coarse-NCL, HD > 1 μm, PDI > 0.6). The formulations were diluted in culture medium (20 μg/1000 μL) at a concentration range of 20–0.08 mg/mL, equivalent to 30–0.12 μM of niclosamide, added to the wells with cells and the plates were incubated for 48 h.

After treatment, the spheroids were analyzed with the CellTiterGlo 3D luminescence kit according to the manufacturer’s instructions. Briefly, 100 μL of the contents of each well were transferred to a flat-bottomed, opaque white 96-well plate along with the spheroid; subsequently, 100 μL of the kit reagent was added to each well. The plate was shaken for 5 min to lyse the spheroids and incubated for 20 min. Readings were performed in an Agilent BioTek Synergy HTX Multi-Mode Microplate Reader at 560 nm.

Data were processed with the Microsoft Excel 365 program. The results were analyzed by a four-parameter nonlinear regression mathematical model with GraphPad Prism 7 software (GraphPad Software, Inc., San Diego, CA, USA) and the graphs were created using Origin 2019.

Results and discussion

Niclosamide differential scanning calorimetry (DSC) and thermogravimetric (TG) analysis

Figure 1 shows DSC (a) and TG (b) curves from niclosamide. The endothermic event in the region 80–100 °C in the DSC curve is possibly attributed to water evaporation of the monohydrate form. The TG curve shows that the moisture degree was ~ 2% due to the percentage of mass reduction at 85 °C, indicating the monohydrate form HA; for HB, water evaporation occurs in the 175–200 °C range. Hence, it is possible that some degree of hydration was present in the sample, which tends to decrease the water solubility from ~ 13 to ~ 1 μg/mL (of the anhydrate and HA monohydrate form, respectively) [25].

Fig. 1
figure 1

Niclosamide DSC (a) and TG (b) curves, and transmittance (%) of the samples according to the respective liquid lipid, during the heating/cooling cycle (c). The objective was to estimate the lipid ability to solubilize niclosamide

Full size image

According to Fig. 1a, the niclosamide melting peak occurred at ~ 228 °C, which is in accordance with the literature [26]. Along with its molecular weight (M.W. 327.12 g/mol), the result is an indication of niclosamide tendency for recrystallization, considering its glass forming ability classification, which is the ability of a material to vitrify on cooling from the melted state, or to form a glass by processes such as spray-drying [27]. In this case, niclosamide is considered a class I molecule, which refers to small (M.M. ~  < 300 g/mol) and stable (melting point > 200 °C) molecules that quickly achieve the required conformation to form the crystal lattice and are highly prone to precipitate into a crystalline form [27, 28].

Liquid lipid and surfactant selection

Figure 1c shows the achieved transmittances (%) of the samples during the heating/cooling cycle in Crystal 16 equipment. The transmittance at the end of the test is in accordance with the visual aspect of the samples since niclosamide was completely dissolved in Capmul® MCM C8, while for the others the drug was mostly suspended or settled in the bottom of the vial (Supplementary material, Fig. S1). The amount of niclosamide used (10 mg/1 g of liquid lipid) was a cautious choice to allow the complete solubilization of the drug. Nevertheless, in most of the lipids niclosamide could not be solubilized (Fig. 1c). This shows that despite the high lipophilicity of drug substances such as niclosamide (logD ~ 4, pH ~ 7, [29]), their solubility in lipidic systems is not straightforward, especially for those presenting high melting points (> 150–200 °C) [28, 30].

Lipids were then grouped according to their estimated ability to solubilize niclosamide (Table 2). The medium-chain Capmul® MCM C8 had the best performance since even at the end of the test, the transmittance was 100%, showing a clear and homogeneous medium. The average group includes medium-chain lipids that despite their capacity to solubilize niclosamide at 80 °C, they were not able to maintain the drug solubilization during the cooling phase. Finally, the group which presented low performance includes long-chain lipids that were not able to solubilize niclosamide at all. These liquid lipids were therefore discarded.

Table 2 Liquid lipid groups according to their estimated ability to solubilize niclosamide (10 mg/g of liquid lipid) during a heating/cooling cycle in Crystal 16 equipment. High: solubilization throughout the running; average: solubilization at 80 °C; low: no solubilization. Number of carbon atoms according to the length of the carbon chain: medium (6–12); long (> 12)
Full size table

Results herein obtained confirmed the hypothesis that medium-chain lipids provided better solubility results for niclosamide than long-chain. The trend that lipids with shorter carbon chains provide better solubility results was observed in other studies [30, 31]. One explanation for this was suggested by Cao and colleagues [32]. Briefly, they proposed that the chain length of the lipids not only influences the hydrophobicity of the triglyceride, but also the molar concentration of the ester group/g of the lipid and its propensity for hydrogen bond interactions with polar/polarizable solutes. Thus, the higher interactions of accepting ester groups in medium-chain triglycerides, compared to long-chain compounds, with the donating polar groups in niclosamide, such as -OH or -NH, could be related to the solubility results herein obtained. We expected that despite the oils sharing most of the fatty acids, different proportions of these components in the oils could influence solubility capacity of the lipid. However, our findings show that long-chain compounds were not able to solubilize niclosamide, independently of the oil composition (Table 2).

We raised the hypothesis that incorporating niclosamide in a lipid-based nanostructured system could allow oral drug delivery via intestinal lymphatic transport. The reason is that metastasis in lymph nodes, and consequently, cell dissemination through lymphatics is closely associated with reduced survival in patients with colon cancer [33]. The absorption through lymphatics is proposed mainly to occur via the chylomicron pathway. In this case, after digestion of the lipid-based formulation, the drug substance and the lipids are assembled into chylomicrons in the enterocytes, which are then secreted to lymphatics. Two factors commonly attributed to favor this phenomenon are the logP of the drug substance (≥ 5) and its lipid solubility (≥ 50 mg/g). However, other factors may also influence chylomicron binding and consequently lymphatics absorption, such as the drug’s pKa, lipid type, and food intake [34, 35].

Considering lipid type, the trend that lipids with a longer chain length favor oral drug absorption via lymphatics has been described in the literature [36, 37]. This may be attributed to the affinity of the fatty acyl-CoA synthetase for long-chain lipids and their activation to form triacylglycerols, with their subsequent assembly into chylomicrons; the low affinity of this enzyme for medium-chain lipids would favor their absorption as soluble fatty acids via the portal vein [38,39,40]. In the case of niclosamide, lipid selection is a limiting factor since long-chain lipids tested here were not able to solubilize the drug substance. However, medium-chain lipids may still be an option for niclosamide lymphatics absorption. For instance, Xia et al. developed a regorafenib self-assembled lipid-based nanocarrier, composed of a mixture of phospholipids with short (glycerol tributyrate) and medium-chain lipids, aimed at targeting the intestinal lymphatic system to treat CRC [41]. Moreover, Caliph et al. detected halofantrine lymphatic transport even using short and medium-chain lipids, which indicates that the use of these components may not be restrictive to achieve lymphatics [36]. Thus, we hypothesize that incorporating niclosamide in lipid-based systems using medium-chain lipids may allow feasible niclosamide oral absorption via the lymphatic transport system.

Because the estimated ability of Capmul® MCM C8 to dissolve niclosamide was high, it was selected to prepare nanoemulsions containing different surfactants (poloxamer 188, Kolliphor® RH 40, Tween™ 80, Gellucire® 44/14). The formulation with poloxamer 188 did not show any signs of phase separation or precipitation. Furthermore, it displayed small particle size (220.4 ± 0.7 nm), low PDI (0.029 ± 0.024), and monomodal distribution, indicating adequate particle size distribution, stabilized by an electrostatic repulsion mechanism (ZP =  − 31.7 ± 0.8). Hence, these results indicated poloxamer 188 as a promising stabilizer for the preparation of the lipid system containing niclosamide.

Preparation of the nanoemulsions

Following liquid lipid and surfactant selections, nanoemulsions containing Capmul® MCM C8 (5–10% w/w), poloxamer 188 (1–3% w/w), and niclosamide (10 mg/1 g of liquid lipid) were prepared. The quality target product profile (QTPP) established for the nanoemulsion was particle size < 300 nm, PDI < 0.2, and agglomerate/aggregate-free preparations determined by visual inspection. This QTPP was chosen to obtain a stable formulation with the smallest particle size possible to improve the bioadhesiveness and uptake by the cells.

Despite the homogeneous appearance, soon after preparation, formulations presented a yellowish precipitate after a short time of storage under refrigeration (4 °C) or at room temperature, and particle size of 0.5–1.0 μm. Thus, niclosamide may not have been encapsulated as we expected; besides, even when encapsulated, its possible expulsion from the nanoparticle may have contributed to the physical instability, considering its strong tendency to recrystallize. To address these issues, some hypotheses were raised and tested. First, we reduced to half the amount of niclosamide, but results were the same after few days (data not shown). Second, we prepared new formulations including surfactants not yet tested (lipoid S100 and Labrasol®) to stabilize the system; and third, we increased the pressure during the high-pressure homogenization procedure. However, formulations still presented phase separation and particle size in the micrometer range.

We then considered another lipid from the liquid lipid selection. According to Table 2, lipids from the average group had equivalent performance on solubilizing niclosamide due to their inability to maintain the drug solubilized at room temperature. We decided to test medium-chain triglyceride Miglyol® 812 since its use to dissolve niclosamide resulted in samples with some transmittance (%) in the 25 °C cooling phase, maintaining the drug:lipid proportion (10 mg:1 g). For this, we evaluated this lipid alone and mixed with Capmul® MCM C8 (50:50). According to the results, formulations containing the lipid mixture still did not meet the QTPP (particle size < 300 nm, PDI < 0.2), presenting poor physical stability (< 7 days). In contrast, formulations containing Miglyol® 812 as the liquid lipid (7.5% w/w), along with poloxamer 188 (3% w/w), met the QTPP, either for the Blank or the niclosamide nanoemulsion, presenting physical stability for at least 10 days.

The combination of Miglyol® 812 and poloxamer 188 in lipid-based systems was used to prepare nanoemulsions containing cannabidiol for colloidal lipid carriers [42]. Furthermore, Real et al. prepared lecithin/span®80-based nanoemulsions with Miglyol® 812, containing model drugs with different logP values (including niclosamide), to assess the influence of factors on the physicochemical properties of the formulations. They found that the Miglyol® 812 proportion and logP of each drug had a strong impact on the stability of the nanoemulsion. In this case, the higher the lipophilicity, the greater the encapsulation efficiency [43]. Therefore, we expected that nanoemulsions with Miglyol® 812, poloxamer 188, and niclosamide would provide viable formulations.

Niclosamide nanoemulsion optimization

Based on our preliminary results, we carried out the nanoemulsion optimization with thirteen formulations from an experimental design (DoE). Considering the challenges in obtaining a stable nanoemulsion, we fixed the niclosamide proportion according to the liquid lipid (10 mg/1 g of liquid lipid). Thus, Table S2 shows size and PDI values from 13 formulations, and Table S3 presents the analysis of variance using particle size as response (Supplementary material). Miglyol® 812 and poloxamer 188 had a significant influence on the response (p-value < α). In addition, lack-of-fit (0.196) indicates that the model is statistically well suited and with significance. Table S4 presents the significance test of the regression coefficients (Supplementary material). Both R2 and Adj R2 values do not differ significantly (0.46%). Furthermore, R2 (pred), R2, and Adj R2 parameters had values greater than 90%, which indicates the probability that the model’s predictions are close to the results found experimentally. Therefore, Eq. (4) is considered adequate to predict the particle size:

$$Size\;\left(nm\right)= 312.7\;+\;7.353\;miglyol\;812-107.5 p188\;+\;16.95\;p188*p188$$
(4)

In this model, poloxamer 188 (P188) has the major contribution to reduce particle size due to its negative coefficient (− 107.5). Miglyol® 812 (7.353) and the quadratic function of the surfactant proportion (16.95) favor the increasing of the particle size. We tested this model with two formulations (A and B), according to Table 3:

Table 3 Tested formulations to verify the mathematic model for prediction of particle size. Components described according to proportion in the formulation (%, w/w), with purified water added to 100%
Full size table

The surface response chart (Fig. 2a) shows the effect of Mygliol® 812 and poloxamer 188 on the response, while the contour plot (Fig. 2b) shows the optimal region for particle size. Values < 200 nm are expected in formulations with poloxamer 188 concentration of at least 2% (w/w) and miglyol® 812 at 5%. However, as the concentration of Miglyol 812 increases, the size tends to increase as well (Fig. 2a).

Fig. 2
figure 2

Surface response (a) and contour plot (b) charts of particle size according to the proportion of Miglyol® 812 and poloxamer 188 in the formulation (% w/w). Stability study of the niclosamide nanoemulsion according to particle size (c) and ZP (d)

Full size image

Particle size, zeta potential, particle concentration, and stability study

Formulation 12 (5.00% Miglyol® 812, 2.25% poloxamer 188, and 0.05% niclosamide, w/w) (Table S2, supplementary material) was selected for the stability study and in vitro performance tests. According to Fig. 2c, particle size ranged from 190 to 217 nm at 30 °C/75% RH, and from 190 to 200 nm at 4 °C, after 56 days of stability. These results suggest that Miglyol® 812 was a suitable choice as the liquid lipid, despite not presenting the highest capacity for drug solubilization. The highly negative ZP (Fig. 2d) contributes to the stability by an electrostatic repulsion mechanism. Average ZP was − 31.5 ± 4.7 and − 29.9 ± 5.7 mV, at 30 °C/75% RH and 4 °C, respectively. Fig. S2 and S3 (supplementary material) show particle size distribution throughout the stability period. It is possible to observe a monomodal distribution for the particle size, with average PDI of 0.14 and 0.15 for samples kept at 30 °C/75% RH and 4 °C, respectively.

Figure S4a (supplementary material) illustrates the opaque appearance of the nanoemulsion. To quantify this characteristic, we estimated the total particle concentration to be 2.79 × 1012 ± 5.98 × 1011 particles/mL. Due to the opacity of the formulation, it was necessary to perform dilutions to obtain readable data. We carefully identified a linear concentration range to ensure accurate measurements (Fig. S4b). This approach is consistent with the method used by Cole and colleagues, who similarly estimated viral concentrations through a series of sample dilutions of recombinant adeno-associated viruses [44].

Encapsulation efficiency and drug loading

After sample preparation and mild centrifugation, we measured the absorbance of the formulation filtrate in absolute ethanol. It was a clear and homogenous solution, which presented negligible reading. Using Eq. (2), we estimated EE (%) as at least ~ 90%, considering the lowest point of the linearity curve; DL (%) was estimated according to Eq. (3) as ~ 0.5 mg/mL. The stability of the nanoemulsion indicated that most of the drug was encapsulated since it presented particle size with monomodal distribution throughout at least 56 days (PDI of ~ 0.15) (Supplementary material). As previously described, using the same amount of niclosamide to prepare nanoemulsions with Capmul® MCM C8, a yellowish precipitate appeared a few days after the preparation, with inadequate particle size (> 1 μm).

Toxicity on Galleria mellonella larvae

According to Fig. 3, the set of larvae that received Nano-NCL presented 75% of survival at day 3, and about 70% at day 5. Those that received the unloaded formulation (Blank) had a survival rate of approximately 55% in the end of experiment. Therefore, results herein obtained indicated that the Galleria mellonella invertebrate living model presented some intolerance to the samples tested, which might be attributed to the presence of Miglyol® 812 and polaxamer 188. Since these excipients are widely used in approved products and described in monographs, we expected that the living model would present better tolerance to the samples. Nevertheless, the toxicity of the nanoemulsion will be monitored in subsequent living model studies.

Fig. 3
figure 3

Toxicity of niclosamide nanoemulsion in the invertebrate model of Galleria mellonella. Survival curve of larvae injected with phosphate buffer solution (PBS), niclosamide nanoemulsion (Nano-NCL, 87.5 mg/kg), and placebo nanoemulsion (Blank)

Full size image

Formulation effects on HCT-116 cell viability

A preliminary stability test was performed to verify whether the nanoemulsion would be able to maintain its size and distribution during the conditions of cell cultivation. Figure 4a shows particle size and PDI of Nano-NCL according to the treatment period in DMEM – F12. As observed, culture media had little influence on the system, evidenced by the stable particle size throughout the treatment period and PDI < 0.2. Figure 4b shows the microscopy image of a well containing 2 mL of a 5 mg/mL Nano-NCL dispersion in DMEM – F12™. Crystallization or precipitates were not observed, which further indicates that niclosamide was mostly encapsulated in the nanoemulsion, and the system was stable during the treatment periods.

Fig. 4
figure 4

Stability of Nano-NCL in DMEM – F12™ culture media: a) particle size (nm, bars) and PDI (line); b) microscopy image of a well containing 2 mL of a 5 mg/mL Nano-NCL dispersion in the culture media. No sign of recrystallization was observed

Full size image

Table 4 shows the IC50 values calculated according to the model and Fig. 5 shows carcinoma cell line HCT-116 cell viability at 24 and 48 h. In summary, niclosamide cytotoxicity was both time and concentration dependent.

Table 4 IC50 values calculated according to the model. Values in milligram per milliliter refer to formulation concentration, considering formulation density as 1 g/mL. Values in μM refer to the drug concentration. NC = not calculated
Full size table
Fig. 5
figure 5

HCT116 cell viability at 24 and 48 h according to concentration of the formulation (a, c) (mg/mL, density = 1 g/mL) or niclosamide (b, d) (μM). Data represented as mean ± standard error of the mean (n = 8–10), in three independent experiments

Full size image

At 24 h, the Blank displayed a dose-dependent viability curve, demonstrating the components of the formulation have cytotoxicity, but IC50 of 15.82 mg/mL is almost three times higher than Nano-NCL (5.842 mg/mL) (Fig. 5a, Table 4), showing that niclosamide is critical for system activity. Moreover, niclosamide incorporation into the nanoemulsion (Nano-NLC) and coarse dispersion promoted a shift of the viability curve to the left compared to the drug solution at 24 h (Fig. 5b), indicating the free drug has lower cytotoxicity and suggesting that the lipid systems potentiated drug cytotoxicity. However, the IC50 values of niclosamide in the Nano-NLC (8.775 μM) and Coarse-NCL (9.936 μM) were very similar, showing the size was not as relevant in this period of treatment.

At 48 h, the Blank nanoemulsion did not show sufficient cytotoxicity to calculate the IC50 value (Table 4), also evidenced by the curve (Fig. 5c), which indicates that niclosamide is essential for the activity of the system. The IC50 value of niclosamide was reduced, demonstrating that its cytotoxicity was time-dependent. Furthermore, the drug IC50 value in Nano-NCL (IC50 = 1.259 μM) was 3.6-fold lower compared to the Coarse-NCL (Fig. 5d), suggesting that, at this time-point, the droplet size might have contributed to potentiate the cytotoxic effect of the drug.

The slight reduction of the cell viability at 24 h (Fig. 5a) might be due to the presence of poloxamer 188 and its capacity to interact with lipid bilayers, changing the microviscosity of cell membranes [45]. Moreover, poloxamers have been described as Pg-p inhibitors [46]. To the best of our knowledge, niclosamide has not been described as a substrate for this pump efflux. In a study with multidrug-resistant cells (overexpressing Pg-p), niclosamide showed toxicity against leukemia cells, probably due to its rapid uptake and Pg-p bypassing, reducing glutathione and promoting elevated reactive oxygen species levels. However, cells from solid tumors, such as breast, lung, and colorectal, were more resistant [47]. Thus, in the case of eventual Pg-p-mediated resistance by CRC cell lines, we hypothesize that the presence of the poloxamer might contribute to niclosamide activity.

In summary, Blank nanoemulsion displayed negligible cytotoxicity and the Coarse-NCL showed almost the same activity when compared to the DMSO-NCL in 48 h. Nano-NCL however demonstrated higher cytotoxicity evidenced by the lower IC50 and left-shifted curves, especially at 48 h, highlighting the importance of the nanometric size to enhance the niclosamide cytotoxicity. It is noticeable that Nano-NCL IC50 in 48 h (IC50 = 1.259 μM) is close to the Osada et al. result [48], when 40% of proliferation for HCT116 was observed at 2 μM of niclosamide in the same treatment period. Inhibition of diverse signaling pathways have been proposed to explain the anticancer activity; among them, the Wnt/β-catenin signaling. Niclosamide actions in this signaling include endocytosis of the Wnt Frizzled receptor, downregulation of disheveled2 and β-catenin expression and inhibition of Tcf/Lef promoter activity, leading to apoptosis in CRC cell models [48, 49].

Formulation effects on the viability of 3D cell culture models

Figure 6 shows the viability of the HCT-116 spheroid cells according to formulation concentration (mg/mL) (a) or niclosamide concentration (μM) (b). The Blank nanoemulsion (Fig. 6a) did not reduce cell spheroid viability, indicating that the components of the formulation have little influence in the concentrations used. DMSO-NCL and Coarse-NCL had similar IC50 values of 1.09 and 1.25 μM, respectively. Nano-NLC displayed a slightly lower IC50 (0.90 μM) value, 1.21 and 1.39-fold lower than DMSO-NCL and Coarse-NCL, respectively. We hypothesize that the molecule could spread through the spheroid even without the nanocarrier, justifying the slight difference among the treatments. Nevertheless, we did not observe lack of efficacy; considering that DMSO is not a safe vehicle to the drug substance, incorporation of niclosamide in the lipid systems represented an advantage.

Fig. 6
figure 6

Viability (%) of the spheroid for 48 h of treatment according to concentration of: a) formulation (mg/mL, density = 1 g/mL); b) niclosamide (μM). Data represented as mean ± standard error of the mean (n = 8–10), in three independent experiments

Full size image

In a tumor microenvironment, nanoparticles have to diffuse in the extracellular matrix (ECM) to reach deeper layers of the spheroid. The high density of cancer cells reduces the ECM space; furthermore, the uncontrolled production of ECM components provides reduced pore size due to fiber cross-linking. Hence, tumor ECM restricts nanoparticle diffusion, and factors such as particle size, ZP, and stiffness may influence their dispersion and cell uptake [50, 51].

For instance, Tchoryk and colleagues developed doxorubicin polymeric nanoparticles of 30, 50, and 100 nm, to evaluate their penetration in HCT116 spheroids. The smaller particles had deeper penetration in the spheroid than the larger, suggesting a size restriction to nanoparticle diffusion. Moreover, unmodified nanoparticles had better performance than those negative or positively charged, indicating an electrostatic mechanism that would hinder their dispersion due to interactions with ECM components [52].

Thus, since nanoparticles herein obtained were at least 200 nm and negatively charged (ZP ≤  − 25 mV), their diffusion may have been restricted in the spheroid. Nevertheless, nanoemulsion performance was higher than the coarse emulsion, indicating that the nanoscale is needed to potentiate the cytotoxic effect of niclosamide. Hence, to further improve IC50, QTPP could include smaller (< 100 nm) and slightly charged nanoparticles. This would be the target for an injectable formulation, directing the nanoparticles to the leaky vessels of the tumor. For oral route administration, nanoparticles are subjected to factors that may compromise their stability, such as pH changes and the presence of digestive enzymes. In this case, lipid-based nanostructured systems aiming at enhanced saturation solubility and absorption via lymphatics seems to be a more promising strategy.

Conclusion

This study aimed at the development, physicochemical characterization, and in vitro anticancer activity of a niclosamide nanoemulsion, with HCT-116 as the cell model. Formulations with Capmul® MCM C8 were not sufficiently stable to justify the usage of this lipid. In contrast, Miglyol® 812 proved to be a suitable liquid lipid for the system. Niclosamide nanoemulsion (~ 200 nm) with Miglyol® 812 and poloxamer 188 was stable for at least 56 days, with monomodal particle size distribution during the period. The cell viability assay with MTT showed that niclosamide efficacy against HCT-116 was both time and concentration dependent, indicating that the nanoscale was fundamental to improve the niclosamide cytotoxic effect. The 3D cell culture model confirmed the monolayer result. Size and surface charge may have influenced the penetration of the nanoparticles in the spheroid. Results herein obtained encourage further research to understand and optimize niclosamide performance as an anticancer drug substance to treat CRC.