Nanotoxicology applied to solid lipid nanoparticles and nanostructured lipid carriers – A systematic review of in vitro data

Purpose: Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) were developed as alternative colloidal carriers with better stability and tolerability than polymeric nanoparticles. However, in vitro toxicity data were scattered across many cell lines, assays, exposure times, concentrations, and formulations, with some contradictory results. A systematic review was needed to consolidate cytotoxicity, oxidative stress, genotoxicity, and hemocompatibility data and identify gaps.

Hypothesis: The authors’ framing thesis: if SLN/NLC are made from biodegradable, generally regarded as safe (GRAS) lipids and suitable surfactants, then they should be well tolerated in vitro, but their toxicity may depend on lipid matrix, surfactant, surface charge, size, cell type, and assay. Systematic analysis of existing data should clarify whether SLN/NLC are truly safe nanocarriers and where evidence is lacking.

Aims: Collect and analyze in vitro data on SLN/NLC cytotoxicity, oxidative stress, genotoxicity, and blood compatibility. - Summarize outcomes by IC50 and % cell viability, with reference tables. - Evaluate the influence of lipid core, surfactant, size, charge, and cell type on toxicity. - Compare SLN/NLC tolerability with other colloidal carriers. - Identify contradictions, methodological issues, and future research needs.

Delivery system: Platform: solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC). - Core materials: solid lipids such as Compritol 888 ATO, Precirol ATO 5, glyceryl monostearate, stearic acid, cetyl palmitate, tripalmitin, Witepsol, Softisan, hydrogenated palm oil; liquid lipids such as oleic acid, Miglyol 812, isopropyl myristate, squalene, soybean oil. - Surfactants/stabilizers: Tween 80, Poloxamer 188, Poloxamer 407, PVA, SDS, lecithin, Solutol HS 15, Cremophor, sodium taurocholate, etc. - Payloads discussed: chemotherapeutic drugs (paclitaxel, doxorubicin, docetaxel, etoposide, mitoxantrone, etc.), gene delivery (plasmid DNA, siRNA), and non-chemotherapeutic drugs. - Targeting/functionalization: some cationic SLN/NLC and surface-modified formulations; not the main focus of this review.

Approach: Systematic review of in vitro studies. PubMed search for “solid lipid nanoparticles” and “cytotoxicity” returned 128 results; ISI Web of Knowledge returned 174 entries (as of December 2013). Studies used many cell lines, including cancer lines (MCF-7, A549, HepG2, HeLa, U87, etc.) and non-cancer lines (NHF, HaCaT, Vero, MDCK, 3T3, granulocytes, macrophages). Exposure times ranged from 4 h to 72 h, with 24 h most common. Concentrations were normalized to mg solid lipid/mL medium where possible. As a review, it reports no primary experimental groups, n values, doses, or controls.

Key methods: No primary methods. The review discusses data generated by cited studies using: - Cell viability assays: MTT, MTS, WST-1, WST-8, Alamar Blue, Neutral Red, Crystal Violet, Trypan Blue, LDH release, propidium iodide. - Oxidative stress: DCFH-DA for ROS, glutathione pool, lipid peroxidation. - Genotoxicity: comet assay. - Hemocompatibility: oxyhemoglobin release/hemolysis assays. - Cellular uptake and phagocytosis assays; uptake mechanism inhibitors. - Particle characterization: size, PDI, zeta potential (from included studies).

Key results: Most reported IC50 values for blank SLN/NLC were in the range 0.1–1.0 mg/mL; some formulations had IC50 >1 mg/mL, and a few were more toxic. - PVA and SDS as surfactants were associated with low IC50 values; Tween 80 formulations were generally well tolerated. - Stearic acid-based SLN caused a drastic decrease to 2.2% cell viability at 1 mg/mL in primary macrophages; other lipids such as Compritol, tripalmitin, and cetyl palmitate caused no cytotoxic effect up to 1 mg/mL in the same study. - SLN/NLC were consistently less toxic than polymeric carriers: polycyanoacrylate nanoparticles at 0.35 mg/mL were 100% lethal to HL-60 macrophages, while SLN at 1.5 mg/mL gave 90% viability; PEI IC50 was 0.009 mg/mL in MDA-MB-435 cells, while SLN showed no toxicity at 0.048 mg/mL. - No clear influence of particle size on toxicity was found from available data; the highest viabilities at high concentrations were observed with SLN between 100–300 nm. - No clear tendency for cancer versus normal cell lines to be more susceptible was observed. - Drug-loaded SLN/NLC usually increased toxicity versus free drug, but some reports showed reduced toxicity, likely due to incomplete drug release. - ROS generation was mostly negligible or low, but one study reported ROS-related viability loss that was partially reversed by antioxidants. - Genotoxicity data were limited; one comet assay study found no genotoxicity risk of cationic SLN. - Hemocompatibility was encouraging: hemolysis was generally <10%, often <5%, and drug-loaded SLN/NLC caused less hemolysis than free drugs. - Uptake was prompt in many cell lines; clathrin-mediated endocytosis appeared preferential, with minor caveolae or macropinocytosis contribution.

Interpretation: The authors conclude that the basic concept of SLN/NLC as well-tolerated carriers is supported, and they appear safer than polymeric nanoparticles. However, contradictory results and methodological heterogeneity mean the overall question of whether SLN/NLC are safe under all circumstances remains unanswered. They call for standardized protocols, more detailed safety endpoints, and in vivo studies that specifically address side effects rather than only efficacy.

Limitations: Systematic review; no primary data, effect sizes, n values, doses, or controls. - High heterogeneity in cell lines, assays, exposure times, concentration reporting, and formulations. - Many studies did not include blank SLN/NLC controls or did not report stability in cell culture media. - IC50 could be calculated in only a few studies. - Lack of standardized reporting and no large series tested in one laboratory. - Limited data on genotoxicity, oxidative stress, and hemocompatibility. - Possible bias because many studies focused on proving efficacy rather than safety. - In vitro–in vivo correlation not established; side effects at administration site and systemic toxicity remain largely unknown. - “Normal” cell lines used are still dividing cells and may not represent mature differentiated tissues.