Impact of solid lipid nanoparticles on 3T3 fibroblasts viability and lipid profile: The effect of curcumin and resveratrol loading

Abstract This study focused on the impact in 3T3 fibroblasts of several types of empty and curcumin‐ and resveratrol‐loaded solid lipid nanoparticles (SLN) on cell viability and lipid metabolism in relation to their lipid content and encapsulated drug. SLN, prepared by hot homogenization/ultrasonication, were characterized with respect to size, polydispersity index, and zeta potential. Compritol® 888 ATO at different concentrations (4%, 5%, and 6% wt/wt) was chosen as lipid matrix while Poloxamer 188 (from 2.2% to 3.3% wt/wt) and Transcutol (TRC; 2% or 4%) were added as nanoparticle excipients. Prepared SLN were able to encapsulate high drug amount (encapsulation efficiency percentage of about 97–99%). All empty SLN did not show cytotoxicity (by MTT assay, at 24 h of incubation) in 3T3 cells independently of the lipid and TRC amount, while a viability reduction in the range 5–11% and 12–27% was observed in 3T3 cells treated with curcumin‐loaded and resveratrol‐loaded SLN, respectively. SLN without TRC did not affect cell lipid metabolism, independently from the lipid content. Empty and loaded SLN formulated with 4% of Compritol and 4% of TRC significantly affected, after 24 h of incubation at the dose of 5 μl/ml, cell polar lipids (phospholipids and free cholesterol) and fatty acid profile, with respect to control cells. Loaded compounds significantly modulated the impact of the corresponding empty formulation on cell lipids. Therefore, the combined impact on lipid metabolism of SLN and loaded drug should be taken in consideration in the evaluation of the toxicity, potential application, and therapeutic effects of new formulations.

on cell homeostasis and physiological functions and represent indispensable substrates for β-oxidation and ATP production Maulucci et al., 2016). Following the biosynthesis, desaturase enzymes introduce a methylene group in saturated FA (SFA) leading to the monounsaturated FA (MUFA), while polyunsaturated FA (n-6 and n-3 PUFA) are produced by elongation and desaturation of the essential linoleic (18:2 n-6) and α-linolenic (18:3 n-3) acids, respectively Maulucci et al., 2016). Free cholesterol, together with phospholipids, is an essential component in the plasma membrane of mammalian cells and plays diverse structural and functional roles (Paukner et al., 2022). Changes in lipid organization (FA and cholesterol) can largely affect membrane functional and biophysical properties (fluidity and lipid rafts organization), severely altering cellular functions such as membrane trafficking, protein dynamics, and signal transduction Maulucci et al., 2016;Paukner et al., 2022;Santos & Schulze, 2012). These membranerelated effects can cause disease in living organisms (Paukner et al., 2022). FA profile has been considered as a homeostatic and metabolic biomarker in normal and pathological cells (Maulucci et al., 2016). Recent studies have proposed FA ratios as a suitable way to efficiently replace the original FA data set for FA metabolism analysis (Graeve & Greenacre, 2020). Cholesterol and/or FA metabolism represent the target for the treatment of several pathological conditions such as metabolic syndrome (Denisenko et al., 2020), diabetes (Langlois et al., 2021), and cancer (Santos & Schulze, 2012). Targeting altered lipid metabolic pathways (FA biosynthesis and desaturation, phospholipids and cholesterol metabolism, and lipid droplet synthesis) has become a promising anticancer strategy (Liu et al., 2017;Rosa et al., 2019;Santos & Schulze, 2012). Moreover FA, in a free form or incorporated into complex lipids, are crucial to proper functions of the epidermis and its appendages (Lin & Khnykin, 2014), and the alteration of skin lipid composition can lead to several dermatological disorders (Drakou et al., 2021). Several phenolic compounds act through the membranes by interacting with membrane lipids and changing the general lipid membrane biophysical properties (structure, organization, fluidity, and packing), membrane dynamics, and the expression of lipogenic enzymes involved in FA metabolism (Kühn et al., 2018;Reis & de Freitas, 2020;Rosa et al., 2020).
Nanoparticles, a class of functional materials with overall dimensions in the nanoscale range, are amply used in pharmacology and medicine (Dhiman et al., 2021;Faraji & Wipf, 2009). Lipid-based nanoparticles (LN), made from biocompatible and biodegradable lipids, are well tolerated in living systems, and represent important systems for enhanced incorporation of hydrophobic compounds into the lipid matrix (Dhiman et al., 2021;Pizzol et al., 2014;Severino et al., 2012).
Plainly, safe, and effective utilization of LN and SLN requires that they do not result in an adverse biological response (Scioli Montoto et al., 2020). LN and SLN cannot be viewed as a simple delivery system but can play an active role in mediating biological effects, affecting viability and cell physiology, in particular lipid metabolism Pitzanti et al., 2020;Rosa et al., 2015). Numerous articles were devoted to exploring the LN and SLN cytotoxic effects (Acevedo-Morantes et al., 2013;Pizzol et al., 2014;Scioli Montoto et al., 2020;Weyenberg et al., 2007) and up-regulation of proinflammatory cytokines (Schöler et al., 2002) in cell systems. Previous studies evidenced the toxicity in mouse 3T3 fibroblasts, J774 macrophages, HaCaT keratinocytes, and red blood cells of SLN formulated with stearic acid (Pizzol et al., 2014;Weyenberg et al., 2007). Remarkably, only a small amount of research was undertaken to investigate the LN and SLN impact on the cell lipid profile (in terms of FA and lipid classes) Pitzanti et al., 2020;Rosa et al., 2015). Previous studies evidenced changes in lipid components (incorporation of oleic acid 18:1 n-9 in the phospholipid and triacylglycerol fractions and lipid droplets accumulation) occurring in human carcinoma HeLa cells when exposed to short-term treatments with monoolein-based cubosomes stabilized by Pluronic F108 Rosa et al., 2015). Moreover, in a preliminary study, we evidenced the effect of SLN loaded with 8-methoxypsoralen in modulating FA and polar lipid profile in fibroblasts, without affecting cell viability (Pitzanti et al., 2020). Therefore, LN should not be considered only as simple carriers for drug delivery, but also as a platform able to modulate specific lipid biosynthetic pathways Rosa et al., 2015). Curcumin (CUR) and resveratrol (RSV) ( Figure 1A) are bioactive liposoluble polyphenolic compounds (Goel et al., 2008;Rosa et al., 2014). The dietary polyphenol CUR has been reported to possess a variety of biological and pharmacological activities (anti-inflammatory, antimicrobial, anticarcinogenic, and antioxidant properties).
The natural polyphenol RSV is consumed worldwide as food items and possesses safety profiles with diverse biological and pharmacological actions, including antioxidation, anti-inflammation, antidiabetic, and anticancer activity (Gomes et al., 2020;Gumireddy et al., 2019).
Because of their low water solubility, several studies have been devoted to improving CUR and RSV bioavailability by incorporation in different formulations (liposomes, nanosuspensions, SLN, and nanostructured lipid carriers) (Gumireddy et al., 2019;Sakellari et al., 2021). Moreover, previous studies evidenced the ability of CUR and RSV to affect cell membrane fluidity and FA metabolism (Kühn et al., 2018;Naeini et al., 2019).
In addition to effects on cell viability, the interaction of SLN with cell lipids is an essential focus in assessing and understanding their toxicity and compatibility, because SLN-induced changes in cell lipids can influence biological membrane properties and affect cellular processes. Starting from all these considerations, the aim of this study was to evaluate the simultaneous impact of different types of unloaded and loaded SLN on cell viability and lipid metabolism in relation to their composition (lipid content, penetration enhancer, and encapsulated compounds). SLN were prepared with different amounts of Compritol ® 888 ATO (monoesters, diesters, and triesters of behenic acid 22:0; the structure of glyceryl behenate is reported in Figure 1A) as lipid matrix, Poloxamer 188 (P188) as nanoparticle stabilizer, and Transcutol ® P (TRC) ( Figure 1A) as penetration enhancer, excipients amply used in cosmetic and pharmaceutical products for their tolerability (Björklund et al., 2016;Cortés et al., 2021;Devi & Agarwal, 2019). In a previous study, we demonstrated that the use of TRC in a SLN formulation containing 4% of Compritol 888 ATO could enhance the cellular uptake of nanoparticles and cell lipid modulation (Pitzanti et al., 2020). CUR and RSV were chosen as loaded compounds for their high liposolubility, bioactivity, and ability to affect lipid metabolism. Prepared SLN were tested in 3T3 fibroblasts, a normal cell line previously used to assess the toxicity and biocompatibility of empty and loaded SLN (Pitzanti et al., 2020;Pizzol et al., 2014;Weyenberg et al., 2007). Moreover, the mouse fibroblast plasma membrane is considered an excellent model system to study how FA influence the membrane (Ibarguren et al., 2014). The effect of unloaded, CUR-loaded, and RSV-loaded   The formulations obtained were stored at 25 C prior to use and characterized 24 h after preparation. The encapsulation efficiency (EE %) of the SLN was calculated using the following equation:

| Cytotoxic activity (MTT assay)
Cytotoxicity of SLN was evaluated in 3T3 fibroblasts by the MTT assay (Pitzanti et al., 2020;Rosa et al., 2015). 3T3 cells were seeded in 96-well plates (density of 3 Â 10 4 cells/well) in 100 μl of serumcontaining media. Experiments were carried out 2 days after seeding (at 90% cell confluence  . Therefore, 24-h incubation time was chosen for successive studies in 3T3 cells.

| Lipid profile modulation in 3T3 cells
3T3 fibroblasts were seeded in T-75 culture flasks at a density of about 10 6 cells/10 ml of complete medium and were used for FA profile modulation experiments at 2 days post-seeding (90% cell confluence). 3T3 fibroblasts were treated with SLN (at a concentration of 5 μl/ml) in fresh medium and incubated at 37 C for 24 h. After treatment, cells were washed with phosphate-buffered saline (PBS), scraped, and centrifuged for 5 min at 1200 g at 4 C. Cell pellets were then separated from supernatants and used for lipid extraction and analyses .

| Extraction and preparation of cell lipid components
Total lipids were extracted from 3T3 cell pellets by the addition of the mixture CHCl 3 /CH 3 OH/H 2 O 2:1:1 as previously reported . Aliquots of the CHCl 3 fraction from each cell sample,  (Rosa et al., , 2019. The Agilent OpenLAB Chromatography data system was used for the recording and integration of the chromatogram data. The identification of cell lipid compounds and CUR was made using standard compounds and conventional UV spectra. Calibration curves of all compounds were constructed using standards and were found to be linear (DAD) and quadratic (ELSD), with correlation coefficients >0.995 (Rosa et al., , 2019. The calibration curve equation, limit of detection (LOD), and correlation coefficient (R 2 ) of the individual FA standards are provided in Table S1.

| Statistical analysis
Data were expressed as a mean ± standard deviation (SD). Evaluation of statistical significance of differences was performed using Graph-

| Effect of SLN formulations loaded with CUR and RSV on 3T3 cell viability and lipid profile
The increase in the percentage amount of the lipid matrix did not induce a significant change in 3T3 cell viability and lipid profile; therefore, the empty-TRC 0%-SLN 4% was then selected as starting formulation, subsequently modified by adding different TRC percentages (Pitzanti et al., 2020)    A preliminary study was performed to determine the CUR absorption in 3T3 cells. Figure S2 shows the chromatographic profiles and

| DISCUSSION
SLN can be used to deliver drugs orally, topically, or via inhalation (Faraji & Wipf, 2009;Battaglia & Ugazio, 2019). The knowledge of SLN interaction with living systems is essential in the perspective of implementing nanotechnologies in a safe way (Severino et al., 2012).
LN are characterized by a high stability and ability to carry hydrophilic and lipophilic compounds in the target organ and are generally well tolerated by the human body, because they are made from physiological compounds leading to the metabolic pathways, minimizing adverse side effects (Musielak et al., 2022;Severino et al., 2012).
In this study, several empty and CUR-or RSV-loaded SLN formulations were physiochemically characterized and tested in 3T3 murine fibroblasts for the evaluation of their impact on cell viability and lipid profile in relation to the lipid content and incorporated compound.
All the empty formulations did not show cytotoxic effect in 3T3 cells at long-term exposure (24 h of incubation), independently of the lipid and TRC amount; therefore, this incubation time was chosen for all cell viability assessments. Components (Compritol ® , TRC and Poloxamer 188) of empty SLN are safe and well-tolerated compounds, used in many cosmetic and pharmaceutical products for skin and/or oral delivery (Björklund et al., 2016;Cortés et al., 2021;Devi & Agarwal, 2019 (Yuan et al., 2008).
In humans, dietary behenic acid is poorly absorbed because of its low bioavailability compared with other FA (Cater & Denke, 2001); therefore, the modulation of lipid profile observed in 3T3 cells after treatment with empty-TRC 4%-SLN 4% is a clear indication of SLN uptake/interaction with plasma membrane (Panariti et al., 2012).