Rifabutin-Loaded Nanostructured Lipid Carriers as a Tool in Oral Anti-Mycobacterial Treatment of Crohn’s Disease

nanomaterials Article Rifabutin-Loaded Nanostructured Lipid Carriers as a Tool in Oral Anti-Mycobacterial Treatment of Crohn’s Disease Helena Rouco 1, Patricia Diaz-Rodriguez 2, Diana P. Gaspar 3, Lídia M. D. Gonçalves 3, Miguel Cuerva 4, Carmen Remuñán-López 5, António J. Almeida 3and Mariana Landin 1,* 1R+D Pharma Group (GI-1645), Strategic Grouping in Materials (AEMAT), Department of Pharmacology, Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, Universidade de Santiago de Compostela-Campus Vida, 15782 Santiago de Compostela, Spain; [email protected] 2Drug Delivery Systems Group, Department of Chemical Engineering and Pharmaceutical Technology, School of Sciences, Universidad de La Laguna (ULL), Campus de Anchieta, 38200 La Laguna (Tenerife), Spain; [email protected] 3Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, 1649-003 Lisbon, Portugal; [email protected] (D.P.G.); [email protected] (L.M.D.G.); [email protected] (A.J.A.) 4 Department of Physical Chemistry, Nanomag laboratory, Universidade de Santiago de Compostela-Campus Vida, 15782 Santiago de Compostela, Spain; [email protected] 5Nanobiofar Group (GI-1643), Department of Pharmacology, Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, Universidade de Santiago de Compostela-Campus Vida, 15782 Santiago de Compostela, Spain; [email protected] *Correspondence: [email protected] Received: 29 September 2020; Accepted: 26 October 2020; Published: 27 October 2020   Abstract: Oral anti-mycobacterial treatment of Crohn’s disease (CD) is limited by the low aqueous solubility of drugs, along with the altered gut conditions of patients, making uncommon their clinical use. Hence, the aim of the present work is focused on the in vitro evaluation of rifabutin (RFB)-loaded Nanostructured lipid carriers (NLC), in order to solve limitations associated to this therapeutic approach. RFB-loaded NLC were prepared by hot homogenization and characterized in terms of size, polydispersity, surface charge, morphology, thermal stability, and drug payload and release. Permeability across Caco-2 cell monolayers and cytotoxicity and uptake in human macrophages was also determined. NLC obtained were nano-sized, monodisperse, negatively charged, and spheroidal-shaped, showing a suitable drug payload and thermal stability. Furthermore, the permeability profile, macrophage uptake and selective intracellular release of RFB-loaded NLC, guarantee an effective drug dose administration to cells. Outcomes suggest that rifabutin-loaded NLC constitute a promising strategy to improve oral anti-mycobacterial therapy in Crohn’s disease. Keywords: rifabutin; nanostructured lipid carriers; cell uptake; Caco-2 cells; oral administration; Crohn’s disease 1. Introduction Crohn’s disease (CD) is a chronic inflammatory bowel condition with a higher predominance in industrialized countries, principally in Western Europe and North America [ 1 ]. The disease is characterized by the presence of outbreaks followed by remission periods [ 1 , 2 ], and although symptomatology is variable, diarrhea, abdominal pain, nausea, vomiting, and weight loss are usually involved [ 1 ]. The inflammatory process is usually transmural, involving any region of the digestive tract, affecting distal ileum and colon mainly [1,2]. Nanomaterials 2020,10, 2138; doi:10.3390/nano10112138 www.mdpi.com/journal/nanomaterials Nanomaterials 2020,10, 2138 2 of 19 CD aetiology has been a controversial topic recently [ 3 ]. Disease development is currently associated with genetic susceptibility and environmental factors, such as alterations in gut microbiome and treatment with antibiotics or non-steroidal anti-inflammatories [ 1 , 2 ]. Nevertheless, it is necessary to highlight the recent increment in scientific literature showing the contribution of the mycobacterial pathogen Mycobacterium avium paratuberculosis (MAP) in CD instauration [ 3 , 4 ]. Moreover, inflamed mucosal and submucosal layers in CD are infiltrated by immune cells such as macrophages [ 5 ]. These cells constitute an interesting target for anti-mycobacterial therapy, since MAP is a facultative intracellular organism that resides in host macrophages, establishing a persistent infection [6]. Despite this information, CD’s current treatment is still focused on the pharmacological control of the inflammatory process (using immunosuppressants, corticosteroids, anti-TNF or anti-interleukin drugs and adhesion molecule inhibitors) with the main objective of maintaining the disease remission stage without the need for surgery [ 1 ]. However, although these treatments improve patients’ quality of life, their ability to modify the long-term evolution of the disease has not been demonstrated yet [ 2 ]. Regarding the antibiotic use in CD, they are nowadays relegated to the treatment of perianal fistulas or disease suppurative complications [ 1 ]. Still, some case reports describe long-term CD remissions after antibiotic therapy [ 4 ]. Moreover, an open label extension phase III study sponsored by RedHill Biopharma is currently actively testing orally administered capsules containing a combination of rifabutin, clofazimine and clarithromycin at fixed doses in CD patients [ 7 ]. This study includes the introduction of a MAP PCR test at the baseline and the evaluation of changes of this blood status during the study [ 7 ], which would give insight into in vivo effectivity of this antibiotic combination [ 8 ] and into the clinical benefit derived from MAP eradication [9]. Although orally administered antimycobacterial drugs constitute a promising strategy in CD treatment, two aspects limit this approach. First, gut physiological parameters are altered in CD patients, which can reduce the possibilities to exploit pH, transit time or microbiome as targeting strategies for drug delivery [ 5 ]. On the other hand, antimycobacterial drugs show high lipophilicity and low oral bioavailability [10–12]. In this context, particulate systems constitute an interesting approach, as they can accumulate in inflamed bowel sites [ 5 ]. Additionally, nanoparticulated systems can be designed to load lipophilic drugs, improving their oral bioavailability [ 13 , 14 ]. Moreover, the drug particle reduction to nano size can lead to an enhanced water solubility and dissolution rate [15]. Among nanoparticulate systems, Nanostructured Lipid Carriers (NLC), the second generation of lipid nanoparticles (LN) [ 16 ], can be good candidates to formulate useful antimycobacterial systems. NLC are solid matrices at both room and body temperatures [ 17 ]. They are composed by a solid lipid and a liquid lipid [ 16 ] and present several advantages over the first generation of LN (known as Solid Lipid Nanoparticles or SLN), such as improved stability, higher suppleness in drug release modulation, and increased drug loading capacity [ 17 ]. NLC “ in vitro ” tolerability seems to be higher in comparison with other colloidal carriers, such as polymeric nanoparticles [ 18 ], making them an interesting option for oral drug administration. Therefore, the aim of this work is to investigate the performance of rifabutin (RFB)-loaded NLC (whose formulation procedure and composition were previously optimized by Artificial Intelligence tools), to demonstrate their safety and suitability to achieve an appropriate intestinal permeability and an efficient macrophages uptake. Our goal is to improve the current Crohn’s disease treatments intended to eradicate MAP housed within intestinal macrophages, an area in which, to the best of our knowledge, nanotechnology has never been applied. In this way, an extensive characterization of the nanosystems in terms of particle size, polydispersity, surface charge, and drug payload, was performed. Thermal resistance, morphology, and drug release from NLC in different simulated media were also evaluated. Furthermore, an analysis of the in vitro performance of NLC in cell cultures including a permeability evaluation through Caco-2 monolayers, along with the assessment of cytotoxicity and uptake in human macrophages, was carried out in order to evaluate the targeting potential of the developed nanocarriers. Nanomaterials 2020,10, 2138 3 of 19 2. Materials and Methods 2.1. Materials Rifabutin (RFB) (98% purity) was purchased from Acros Organics ™ (Fair Lawn, NJ, USA). Polysorbate 80 (Tween ® 80), Coumarin 6, dialysis membrane (Spectrum ™ Labs Spectra/Por, MWCO 3.5 KDa), and phorbol 12-myristate 13-acetate (PMA) were acquired from Sigma Aldrich (St Louis, MO, USA). Oleic acid was obtained from Merck (Darmstadt, Germany). Precirol ® ATO 5 (glyceryl distearate) and Epikuron ® 145 V (deoiled phosphatidyl choline-enriched lecithin) were kind gifts from Gattefoss é (Saint-Priest, France) and Cargill (Wayzata, MN, USA) respectively. THP-1, Caco-2 human colon carcinoma and RAW 264.7 cell lines were obtained from ATCC (Manassas, VA, USA). Alexa Fluor ™ 647 phalloidin, ProLong ® Gold Antifade reagent with DAPI, Gibco ™ antibiotic-antimycotic (amphotericin B, penicillin, streptomycin), trypsin-EDTA, foetal bovine serum (FBS), Roswell Park Memorial Institute 1640 Medium (RPMI 1640), Minimum Essential Media ( α -MEM), and phosphate buffered saline (PBS) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Dulbeco’s Modified Eagle Medium (DMEM) was purchased from Corning (Corning, NY, USA). Antibiotic solution (10.000 units/mL penicillin, 10.000 µ g/mL streptomycin) was acquired from GE Healthcare Life Sciences (Chicago, IL, USA). Cell proliferation kit (WST-1) was purchased from Roche (Basel, Switzerland). Ultrapure water (MilliQ plus, Millipore Ib é rica, Madrid, Spain) was used throughout and the remaining solvents and reagents were analytical or HPLC grade. 2.2. NLC Formulation Several batches of NLC loaded with RFB were developed utilizing the composition and operating conditions of a previously optimized NLC system using Artificial Intelligence tools [ 19 ]. Briefly, the components of the formulation were Precirol ® ATO 5 and oleic acid as the lipid components (25:75 ratio), and Tween ® 80 and Epikuron ® 145 V as surfactants. The drug (15 mg) was dissolved in the molten lipid phase at 80 ◦ C (300 mg). The aqueous phase (10 mL), a dispersion of Epikuron ® 145 V (0.5% w/wregarding the lipid phase weight) and Tween ® 80 (1.9% w/vregarding aqueous phase) in Milli-Q ® water, was heated at the same temperature, added to the lipid phase and hot shear homogenized (80 ◦ C) using an Ultra-Turrax T25 (IKA Labortechnik, Staufen, Germany) at 14,800 rpm for 10 min, in a water bath. NLC dispersions were rapidly cooled by transferring them to an ice bath, with gentle stirring, for 2 min. Formulations were carried out in quintuplicate and subsequently dialyzed overnight (MWCO 3.5 KDa), in order to remove the non-incorporated components and obtain the purified NLC. 2.3. NLC Characterization 2.3.1. Particle Size, Surface Charge and Physical Stability Particle size, polydispersity index and surface charge of NLC were determined using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). For size and polydispersity index determinations, samples were placed in polystyrene cuvettes and diluted with Milli-Q ® water (1:10). Surface charge was determined as zeta potential through particle mobility in an electric field. To carry out this determination, samples were also diluted with Milli-Q ® water (1:10) and placed in a specific cuvette where a potential of ± 150 mV was established. All the measurements were performed at 25 ± 1 ◦ C by quadruplicate. 2.3.2. Transmission Electron Microscopy (TEM) Transmission electron microscopy was employed to evaluate morphology of blank (control NLC without drug) and RFB-loaded NLC and to confirm particle sizes previously obtained by DLS. Thus, NLC suspensions were placed on formvar/carbon-coated grids (400 mesh) and stained with 2% (w/v) uranyl acetate. Finally, samples were analysed using a JEOL microscope (JEM 1010, Tokyo, Japan). Nanomaterials 2020,10, 2138 4 of 19 Images were then obtained by using a CCD Orius-Digital Montage Plug-in camera (Gatan, Inc., Pleasanton, CA, USA) and analysed by means of a Gatan Digital Micrograph software (Gatan, Inc., USA). The number of particles considered for size determinations were 44 and 12 for blank and loaded NLC, respectively. 2.3.3. Atomic Force Microscopy (AFM) NLC morphology, particle size and size distribution were also analysed by atomic force microscopy (AFM). This technique is based on the electrostatic interaction between the sample and the AFM tip, which allows for the determination of a sample topography. Measurements were conducted under normal ambient conditions using an XE-100 instrument (Park Systems, Suwon, South Korea) in non-contact mode with the high-resonance non-contact AFM cantilever (ACTA probe, n=330 kHz ). For AFM imaging, 20 µ L of the sample were dropped onto freshly exfoliated mica sheet (SPI Supplies, grade V-1 Muscovite) and after 5 min the mica was washed with Milli-Q water and dried under nitrogen flow. All experiments were performed at room temperature. XEI ® data processing tool (Park Systems, South Korea) were used for the analysis of the obtained data, which were adjusted to a gaussian distribution. 2.3.4. Encapsulation Efficiency and Drug Loading Encapsulation efficiency and drug loading determinations were performed as previously described [ 19 ]. Purified NLC and non-purified NLC (200 µ L) were dissolved with acetonitrile (1.5 mL) and centrifuged at 16,099 × gand 4 ◦ C for 30 min. Centrifugation produces the precipitation of the lipid phase, while the drug remains in the supernatant. RFB quantification was performed by High Performance Liquid Chromatography (HPLC) as described in Section 2.4. The amount of drug quantified in the supernatant of non-purified nanoparticles was used as total drug content. Encapsulation efficiency (EE) and drug loading (DL) of NLC were calculated using the following equations: EE (%) =[(Wloaded drug)/Wtotal drug]×100, (1) DL (%) =[(Wloaded drug)/Wlipid]×100, (2) where W loaded drug is the amount of drug successfully incorporated in the formulation (remaining in the supernatant following acetonitrile addition), W total drug is the total amount of drug, and W lipid is the weight of the lipid vehicle. 2.3.5. Thermal Analysis Using Dynamic Light Scattering (DLS) The influence of temperature on both blank and RFB-loaded NLC suspensions stability was analysed by DLS in a Zetasizer Nano ZS. Three batches of each type of NLC (blank and loaded with RFB) were diluted as described above, and particle size measurements were made during heating and cooling cycles (25 ◦ C-90 ◦ C-25 ◦ C) at 0.5 ◦ C/min in quartz cells. Particle size determinations were recorded every 0.5 ◦C. Each batch was analysed in duplicate. 2.3.6. In Vitro Release Studies RFB release from loaded NLC was investigated in simulated intestinal fluid (SIF) and macrophage’s lysate, in order to compare NLC behaviour in different environments, the intestinal tract and inside macrophages. SIF with pancreatin was prepared according to United States Pharmacopeia (USP). In order to obtain macrophages cell lysate, Raw 264.7 cells (a murine macrophage cell line) were cultured in DMEM supplemented with 10% foetal bovine serum (FBS) and 1% penicillin/streptomycin and incubated at 37 ◦ C and 5% CO 2 . Cells were split when reaching 80% confluence by trypsinization and expanded until achieving enough number of cells. Cells were then trypsinized using standard conditions, washed with PBS, centrifuged, and resuspended in Milli-Q ® water in order to achieve a Nanomaterials 2020,10, 2138 5 of 19 concentration of 3.125 million cells/mL. Cell lysis was performed by subjecting the cell suspension to three freeze-thaw cycles. Drug release studies were performed by quadruplicate at 37 ◦ C in horizontal Franz diffusion cells, where a 1:3 dilution of the nanoparticle suspension in release medium was put in the donor chamber. A dialysis membrane (MWCO 3.5 KDa) was placed between the two chambers in order to avoid the presence of NLC in the receptor chamber. At pre-set times, samples were taken from the receptor chamber and replaced with fresh medium. Drug quantification was performed by HPLC. 2.4. High Performance Liquid Chromatography Method RFB was quantified following a validated method previously described [ 20 ], using an Agilent 1100 HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a C18 column (Waters symmetry 5 µm, 3.9 ×150 mm). Throughout HPLC analysis, 20 µL of each sample were injected and eluted with a mobile phase composed by a mixture of sodium acetate 0.05 M/potassium dihydrogen phosphate 0.05 M (pH adjusted to 4.0 with acetic acid) and acetonitrile (Scharlau, Barcelona, Spain) in a 53:47 (v/v) proportion. Drug quantification was performed at 275 nm, with a 1 mL/min flow rate in an isocratic mode. 2.5. In Vitro Cell Studies 2.5.1. Cell Viability Studies Cytotoxicity of NLC formulations was analysed using WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2Htetrazolium, monosodium salt; Roche, Indianapolis, IN, USA), which produces a water-soluble formazan dye upon cellular reduction by the mitochondrial succinate-tetrazolium reductase [ 21 , 22 ]. Human monocytes (THP-1) were cultured in RPMI 1640 supplemented with 10% heat-inactivated foetal bovine serum (FBS), 1% penicillin/streptomycin and 2-mercaptoethanol 0.05 mM at 37 ◦ C and 5% CO 2 . Five days before the experiment, cells were differentiated to macrophages by stimulation with 200 nM of PMA (Phorbol 12-myristate 13-acetate) for 3 days at a cell density of 2 × 10 5 cells/mL. Then, PMA-containing medium was replaced by fresh medium and cells were incubated for another 2 days with normal media. The day before the experiment, cells were seeded at a density of 2.5 × 10 4 cells/well in 96-well plates. Purified NLC samples were diluted to achieve a final concentration of 0.3, 0.12, 0.06, and 0.03 mg/mL of nanoparticles solid mass per volume. To evaluate cell viability, macrophages were incubated with blank and RFB-loaded NLC formulations, as well as with RFB solutions (concentration equivalent to those present in the previous NLC dilutions), for 24 h (37 ◦ C, 5% CO 2 ). After the incubation period, samples were removed and 10 µ L of WST-1 reagent along with 100 µ L of culture medium were added to each well. After 2 h of incubation with WST-1 reagent, absorbance was read at 450 nm in a microplate reader (Model 680, Bio-Rad, Hercules, CA, USA). Cell viability relative to negative control (Milli-Q®water or DMSO, as appropriate) was calculated according to the following equation: Cell viability (%) =(Sample Absorbance/Control Absorbance) ×100, (3) 2.5.2. Confocal Microscopy Qualitative analysis of NLC internalization by THP-1 derived macrophages was performed by confocal microscopy. For this purpose, nanoparticles were fluorescently labelled with coumarin 6 by incorporating the fluorophore into the oil phase during the formulation process. Cells were seeded at a concentration of 5.3 × 10 4 cells/cm 2 in chambered cell culture slides (Nunc ™ Lab-Tek II Chamber Slide ™ , Thermo Fisher Scientific, Waltham, MA, USA) the day before the experiment. Then, cells were incubated at 37 ◦ C and 5% CO 2 for 5 h with the samples (blank and RFB-loaded NLC), which were added in a final concentration of 0.12 mg/mL. After this incubation period, culture medium was removed, and cells were washed twice with pre-warmed phosphate buffered saline (PBS). Cell fixation Nanomaterials 2020,10, 2138 6 of 19 was performed using a 3.7% formaldehyde solution in PBS for 10 min at room temperature, followed by two washing steps with PBS. Then, a 0.1% Triton X-100 solution was added to permeabilize the cell membrane. Finally, cells were incubated with a 1:40 dilution of Alexa Fluor ™ 647 phalloidin in PBS for 20 min in order to label the macrophages cytoskeleton, and after two extra washing steps, macrophages nucleus were stained with ProLong ® Gold Antifade reagent with DAPI. Images were obtained using a confocal laser microscopy Leica SP5 (Leica Microsystems, Wetzlar, Germany). 2.5.3. Macrophage Uptake Quantification To quantify NLC uptake by THP-1 derived macrophages, NLC were fluorescently labelled with coumarin 6 as previously described. Dialysis of the samples was also accomplished prior to performing the experiment. Macrophage uptake quantification was carried out according to a method previously described [ 23 ] with slight modifications. First, macrophages were seeded in 96-well plates at a cell density of 2.5 × 10 5 cells/mL and 100 µ L per well; nanoparticle suspensions were added to them at a final concentration of 0.12 mg/mL, and fluorescence was determined in a microplate reader (Fluostar Optima, BMG Labtech, Offenburg, Germany) at an excitation and emission wavelength of 485 and 520 nm, respectively (Initial fluorescence). Cells were then incubated during 2 h at 37 ◦ C and 5% CO 2 . Samples were removed, and cells were subjected to three washing steps with 250 µ L of a 20 mM glycine solution in PBS pH 7.4, in order to remove non-internalized nanoparticles and to quench their fluorescent signals. Finally, 100 µ L of Triton X-100 1% were added to disrupt cellular membrane, and fluorescence was again measured (Fluorescence post-lysis). Macrophage uptake was calculated according to the following equation: Macrophage uptake (%) =(Fluorescence post-lysis/Initial fluorescence) ×100, (4) 2.5.4. Nanoparticle Permeation across Caco-2 Cells Monolayers Permeation studies were performed in human colon carcinoma Caco-2 cell line according to a previously described protocol [ 24 ], with modifications. Cells were seeded at a concentration of 6.25 ×103cells/cm2 in Corning ® Transwell ® polycarbonate membrane cell culture inserts (Corning, Corning, NY, USA) and cultured in α -MEM supplemented with 20% FBS, 1% penicillin/streptomycin and 1% antibiotic/antimycotic. Culture medium was replaced every 3–4 days and cells were incubated at 37 ◦ C and 5% CO 2 for 28 days, approximately, until the monolayer reached a suitable transepithelial electrical resistance (TEER). At the beginning of the experiment, TEER was higher than 400 Ω cm 2 , which indicates the formation of an intact monolayer [24]. RFB-loaded NLC fluorescently labelled with coumarin 6 at a final concentration of 0.12 mg/mL or pure Milli-Q ® water (control), were added in the donor compartment. After 2, 4, 6, 24, and 48 h, samples were taken from the receptor compartment and replaced by fresh medium. Fluorescence was measured in a microplate reader (Fluostar Optima, BMG Labtech, Germany), as previously described, in order to evaluate NLC passage across the cell monolayer. Moreover, in order to correlate the amount of NLC present in the receptor compartment with the fluorescent signal obtained, a calibration curve was prepared in triplicate by measuring the fluorescence of known amounts of coumarin 6-labelled RFB-loaded NLC. Finally, permeability of NLC across Caco-2 cells was expressed either as the concentration of permeated RFB ( µ g/mL) regarding time elapsed or as a function of the apparent permeability coefficient (P app ), which is employed to investigate the transport rate. P app was determined according to the following equation: Papp (cm/s) =dQ/dt ×1/(A ×C0), (5) where C 0 is the initial RFB concentration in the upper compartment (6 µ g/mL), A is the growth area (0.33 cm 2 ) and dQ/dt is the appearance rate of the particles on the lower chamber based on its Nanomaterials 2020,10, 2138 7 of 19 cumulative transport for 48 h. This linear appearance rate was calculated as the slope resulting from the representation of the RFB amount present in the receptor compartment versus time. 2.6. Statistical Analysis All experiments were performed at least in triplicate. The data were expressed by mean ± SD and treated with IBM SPSS 24 software. The confidence interval was 95% (p ≤ 0.05). The groups were compared by performing one-way or two-way analysis of variance (ANOVA), as appropriate, followed by post hoc Tukey’s Multiple Comparison Test, and the significant differences between groups were determined. 3. Results and Discussion 3.1. NLC Characterization NLC formulation procedure and composition were beforehand optimized by Artificial Intelligence (AI) tools in order to achieve optimal physicochemical properties along with a suitable drug payload [ 19 ]. Stability of the developed nanocarriers has proven to be adequate after 1 month of storage at 5±1◦C , in terms of particle size, polydispersity index and drug payload. Minor changes without impact over colloidal stability were found for zeta potential [ 19 ]. Besides, an estimation of the characteristics of RFB-loaded NLC, prepared with these optimized parameters, was also provided [ 19 ]. In this way, to verify the robustness of this optimizations process, RFB-loaded NLC were prepared, and particle size, PDI, ZP, and drug payload were again determined. Furthermore, this work includes further characterization of these nanocarriers in terms of morphology, thermal behaviour, release profile, and in vitro performance in cell cultures. 3.1.1. Particle Size, Surface Charge, Physical Stability, and Drug Payload Blank and RFB-loaded NLC were prepared using hot high shear homogenization. Formulations were carried out in quintuplicate, dialyzed overnight and fully characterized in terms of particle size, size distribution, surface charge, and drug load (Table 1). Table 1. Blank and RFB-loaded NLC characterization in terms of particle size, PDI, ZP, EE, and DL (n =3±SD). NLC Size (nm) PDI ZP (mV) EE (%) DL (%) Blank 111 ±3 0.23 ±0−26 ±2 - - RFB-loaded 151 ±34 0.22 ±0.02 −24 ±2 92.83 ±3.75 4.62 ±0.33 Particle size and size distribution are known to affect NLC characteristics such as stability, release rate and biologic performance [ 17 ], and because of that, they should be carefully characterized. NLC formulations showed particle sizes within the nano range, with values of 111 ± 3 nm and 151 ±34 nm , for blank and RFB-loaded nanocarriers, respectively. Differences in size observed between blank and loaded formulations could be associated with the required accommodation space for the drug [ 25 ]. Regarding particle size distribution, blank NLC showed a polydispersity index (PDI) value of 0.23 ±0.00 , whereas the loaded ones displayed an almost identical value of 0.22 ± 0.02. Therefore, the obtained PDI values were below 0.3 in both cases, which is an acceptable value for lipid nanocarriers and indicative of homogeneous particle size distribution [ 26 ]. Remarkably, both size and PDI values obtained for RFB-loaded NLC are in close agreement with those previously predicted by Artificial Intelligence tools, which have been reported to be 152 nm and 0.23, for size and PDI, respectively [ 19 ]. Moreover, both blank and loaded nanocarriers showed zeta potential values close to − 25 mV ( − 26 ± 2 and − 24 ± 2 mV, for blank and RFB-loaded NLC, respectively), which guarantees a good colloidal stability if emulsifiers are included among formulation components [ 17 , 27 ]. These results differ slightly from those predicted by Artificial Intelligence tools, which showed slightly less negative Nanomaterials 2020,10, 2138 8 of 19 values ( − 19 mV) [ 19 ]. However, these differences in ZP could be easily associated with the dialysis step performed in this work after NLC formulation, which can favour the removal of NLC superficial components, such as Tween ® 80, a non-ionic emulsifier. Since this type of emulsifier has been reported to localize close to the nanoparticle interface, counteracting the negative charge of the lipid matrix [ 28 ], its partial removal is expected to lead to a more negative zeta potential. Furthermore, these small differences could only have a slight impact on colloidal stability and are not likely to influence the in vivo fate of the nano-formulations. Concerning drug payload, RFB was incorporated to NLC at 5% (w/w) regarding lipid matrix weight showing a suitable drug payload, with an encapsulation efficiency (EE) of 92.83 ± 3.75% and a drug loading (DL) of 4.62 ± 0.33%. These values suggest that almost all the added drug was successfully incorporated into the nanoparticle matrix. In the same way as in the case of particle size and PDI, EE and DL values obtained are almost identical to those predicted by Artificial intelligence tools, which have been reported to exhibit values of 100% and 5%, for EE and DL, respectively [19]. Hence, the NLC physicochemical characterization data show that they have a particle size within the nano-range, a monodisperse particle size distribution and a suitable drug payload. Besides, the highly negative zeta potential exhibited by the formulations is expected to promote a good colloidal stability. Finally, the results of RFB-loaded NLC characterization closely agree with those predicted by Artificial Intelligence, demonstrating the suitability of these tools to successfully optimize the design of nanoparticle-based drug delivery systems and develop robust and reproducible protocols of NLC preparation. 3.1.2. Thermal Analysis Using Dynamic Light Scattering (DLS) To assess NLC thermal stability, blank and RFB-loaded formulations were subjected to a heating stage from 25 ◦ C to 90 ◦ C, followed by a cooling step to the initial temperature. This approach was previously described to investigate the ability of lipid nanoparticle formulations to maintain their initial properties during high temperature-related procedures [23,29]. In the case of blank NLC (Figure 1A), particle size remains almost unchanged during the whole thermal analysis. A similar behavior was observed for RFB-loaded NLC (Figure 1B) but showing a slight reduction in nanoparticle size. Particle size maintenance along with the negligible size variations exhibited by both formulations throughout the assay indicate a good thermal stability. Therefore, results obtained suggest the developed NLC formulations are suitable for further temperature-requiring processes, as is the case of spray-drying [ 23 ], that could simplify the oral administration of NLC obtaining dried powders, which can be easily administered in capsules or tablets [30]. Nanomaterials 2020, 10, x FOR PEER REVIEW 8 of 18 successfully incorporated into the nanoparticle matrix. In the same way as in the case of particle size and PDI, EE and DL values obtained are almost identical to those predicted by Artificial intelligence tools, which have been reported to exhibit values of 100% and 5%, for EE and DL, respectively [19]. Hence, the NLC physicochemical characterization data show that they have a particle size within the nano-range, a monodisperse particle size distribution and a suitable drug payload. Besides, the highly negative zeta potential exhibited by the formulations is expected to promote a good colloidal stability. Finally, the results of RFB-loaded NLC characterization closely agree with those predicted by Artificial Intelligence, demonstrating the suitability of these tools to successfully optimize the design of nanoparticle-based drug delivery systems and develop robust and reproducible protocols of NLC preparation. Table 1. Blank and RFB-loaded NLC characterization in terms of particle size, PDI, ZP, EE, and DL (n = 3 ± SD). NLC Size (nm) PDI ZP (mV) EE (%) DL (%) Blank 111 ± 3 0.23 ± 0 −26 ± 2 - - RFB-loaded 151 ± 34 0.22 ± 0.02 −24 ± 2 92.83 ± 3.75 4.62 ± 0.33 3.1.2. Thermal Analysis Using Dynamic Light Scattering (DLS) To assess NLC thermal stability, blank and RFB-loaded formulations were subjected to a heating stage from 25 °C to 90 °C, followed by a cooling step to the initial temperature. This approach was previously described to investigate the ability of lipid nanoparticle formulations to maintain their initial properties during high temperature-related procedures [23,29]. In the case of blank NLC (Figure 1A), particle size remains almost unchanged during the whole thermal analysis. A similar behavior was observed for RFB-loaded NLC (Figure 1B) but showing a slight reduction in nanoparticle size. Particle size maintenance along with the negligible size variations exhibited by both formulations throughout the assay indicate a good thermal stability. Therefore, results obtained suggest the developed NLC formulations are suitable for further temperature-requiring processes, as is the case of spray-drying [23], that could simplify the oral administration of NLC obtaining dried powders, which can be easily administered in capsules or tablets [30]. (A) (B) Figure 1. Dynamic light scattering thermograms of (A) Blank NLC formulations and (B) RFB-loaded NLC formulations. 3.1.3. Transmission Electron Microscopy (TEM) TEM technique was employed to evaluate both blank and RFB-loaded NLC morphology as well as to verify nanocarriers size, as recommended elsewhere [27]. As shown in Figure 2, NLC exhibit a spheroidal morphology. Furthermore, in some images (such as Figure 2B), a structure with concentric layers could be noticed, which is also disturbed towards the center of the nanoparticle, exhibiting a high electron density. This lipid nanoparticle structure has been previously described and is Figure 1. Dynamic light scattering thermograms of ( A ) Blank NLC formulations and ( B ) RFB-loaded NLC formulations. Nanomaterials 2020,10, 2138 9 of 19 3.1.3. Transmission Electron Microscopy (TEM) TEM technique was employed to evaluate both blank and RFB-loaded NLC morphology as well as to verify nanocarriers size, as recommended elsewhere [ 27 ]. As shown in Figure 2, NLC exhibit a spheroidal morphology. Furthermore, in some images (such as Figure 2B), a structure with concentric layers could be noticed, which is also disturbed towards the center of the nanoparticle, exhibiting a high electron density. This lipid nanoparticle structure has been previously described and is associated with the polymorphic α -form of lipids [ 31 ]. Moreover, a size of 119 ± 41 nm in the case of blank NLC and slightly higher (173 ± 85 nm) in the case of RFB-loaded ones was observed, confirming the results obtained by DLS. Nanomaterials 2020, 10, x FOR PEER REVIEW 9 of 18 associated with the polymorphic α-form of lipids [31]. Moreover, a size of 119 ± 41 nm in the case of blank NLC and slightly higher (173 ± 85 nm) in the case of RFB-loaded ones was observed, confirming the results obtained by DLS. (A) (B) Figure 2. Transmission electron micrographs of (A) Blank and (B) RFB-loaded NLC. 3.1.4. Atomic Force Microscopy (AFM) Blank and RFB-loaded NLC morphology, particle size and distribution were also assessed by Atomic force microscopy, a technique which gives insight into the sample z-dimension from the deflection of a fine leaf spring (known as the AFM cantilever) [32]. Therefore, AFM is a useful tool to complete the information obtained by DLS and by the two-dimensional images provided by TEM. In this way, the AFM images of blank and RFB-loaded nanoparticles depicted in Figure 3 confirm the spheroidal shape previously shown by TEM. Moreover, results derived from AFM analysis were expressed as the frequency (%) of nanoparticles exhibiting a specific height. Thus, blank and RFB- loaded nanoparticles exhibited a similar size, as values of 43 ± 3 nm and 33 ± 1 nm, respectively, were obtained (Figure 4). The smaller nanoparticle height reported by AFM in comparison with the diameters obtained by DLS and TEM corroborates the existence of a spheroidal structure, closer to a disk than to a sphere. This structure further confirms the prevalence of the polymorphic α-form of lipids [27,31], as previously mentioned, which has been associated with a high loading capacity and a low tendency to expulse the encapsulated drug from the lipid matrix [27]. (A) (B) Figure 3. Atomic force microscopy images of (A) Blank and (B) RFB-loaded NLC. Figure 2. Transmission electron micrographs of (A) Blank and (B) RFB-loaded NLC. 3.1.4. Atomic Force Microscopy (AFM) Blank and RFB-loaded NLC morphology, particle size and distribution were also assessed by Atomic force microscopy, a technique which gives insight into the sample z-dimension from the deflection of a fine leaf spring (known as the AFM cantilever) [ 32 ]. Therefore, AFM is a useful tool to complete the information obtained by DLS and by the two-dimensional images provided by TEM. In this way, the AFM images of blank and RFB-loaded nanoparticles depicted in Figure 3confirm the spheroidal shape previously shown by TEM. Moreover, results derived from AFM analysis were expressed as the frequency (%) of nanoparticles exhibiting a specific height. Thus, blank and RFB-loaded nanoparticles exhibited a similar size, as values of 43 ± 3 nm and 33 ± 1 nm, respectively, were obtained (Figure 4). The smaller nanoparticle height reported by AFM in comparison with the diameters obtained by DLS and TEM corroborates the existence of a spheroidal structure, closer to a disk than to a sphere. This structure further confirms the prevalence of the polymorphic α -form of lipids [ 27 , 31 ], as previously mentioned, which has been associated with a high loading capacity and a low tendency to expulse the encapsulated drug from the lipid matrix [27]. 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