Solid lipid nanoparticles for thermoresponsive targeting: evidence from spectrophotometry, electrochemical, and cytotoxicity studies

Abstract

Thermoresponsive drug delivery systems are designed for the controlled and targeted release of therapeutic payload. These systems exploit hyperthermic temperatures (>39°C), which may be applied by some external means or due to an encountered symptom in inflammatory diseases such as cancer and arthritis. The objective of this paper was to provide some solid evidence in support of the hypothesis that solid lipid nanoparticles (SLNs) can be used for thermoresponsive targeting by undergoing solid-liquid phase transition at their melting point (MP). Thermoresponsive lipid mixtures were prepared by mixing solid and liquid natural fatty acids, and their MP was measured by differential scanning calorimetry (DSC). SLNs (MP 39°C) containing 5-fluorouracil (5-FU) were synthesized by hot melt encapsulation method, and were found to have spherical shape (transmission electron microscopy studies), desirable size (<200 nm), and enhanced physicochemical stability (Fourier transform infrared spectroscopy analysis). We observed a sustained release pattern (22%-34%) at 37°C (5 hours). On the other hand, >90% drug was released at 39°C after 5 hours, suggesting that the SLNs show thermoresponsive drug release, thus confirming our hypothesis. Drug release from SLNs at 39°C was similar to oleic acid and linoleic acid nanoemulsions used in this study, which further confirmed that thermoresponsive drug release is due to solid-liquid phase transition. Next, a differential pulse voltammetry-based electrochemical chemical detection method was developed for quick and real-time analysis of 5-FU release, which also confirmed thermoresponsive drug release behavior of SLNs. Blank SLNs were found to be biocompatible with human gingival fibroblast cells, although 5-FU-loaded SLNs showed some cytotoxicity after 24 hours. 5-FU-loaded SLNs showed thermoresponsive cytotoxicity to breast cancer cells (MDA-MB-231) as cytotoxicity was higher at 39°C (cell viability 72%-78%) compared to 37°C (cell viability >90%) within 1 hour. In conclusion, this study presents SLNs as a safe, simple, and effective platform for thermoresponsive targeting.

Keywords: 5-fluorouracil; breast cancer; emulsions; fatty acids; nanostructured lipid carriers; temperature sensitive.

Figure 1

Schematic presentation of the DPV method for drug release studies. Notes: 5-FU released from SLNs will undergo oxidation due to applied pulsed voltage. The oxidized 5-FU is accumulated at the working electrode and produces signal proportional to concentration of 5-FU in the dissolution medium. Abbreviations: DPV, differential pulse voltammetry; 5-FU, 5-fluorouracil; SLN, solid lipid nanoparticle.

Figure 2

Preparation of TLMs and their characterization. Note: (A) Standard curve of lipid mixtures LO and LL for selections of lipid mixture with MP of 39°C, (B) light transmittance from lipid mixtures LO and LL for solid–liquid phase transition, and (C) light transmittance of SLNs prepared from LO (FLOM and FLOD) and LL (FLLD). Abbreviations: TLM, thermoresponsive lipid mixture; LO, lauric acid and oleic acid; LL, lauric acid and linoleic acid; MP, melting point; SLN, solid lipid nanoparticle; 5-FU, 5-fluorouracil; FLOM, 5-FU-loaded lauric acid and oleic acid nanoparticles prepared with mono-surfactant system; FLOD, 5-FU-loaded lauric acid and oleic acid nanoparticles prepared with double-surfactant system; FLLD, 5-FU-loaded lauric acid and linoleic acid nanoparticles prepared with double-surfactant system; SLN, solid lipid nanoparticle.

Figure 3

TEM images of (A) FLOM, (B) FLOD, (C) FLLM and (D) FLLD. Abbreviations: TEM, transmission electron microscopy; FLOM, 5-FU-loaded lauric acid and oleic acid nanoparticles prepared with mono-surfactant system; FLOD, 5-FU-loaded lauric acid and oleic acid nanoparticles prepared with double-surfactant system; FLLM, 5-FU-loaded lauric acid and linoleic acid nanoparticles prepared with mono-surfactant system; FLLD, 5-FU-loaded lauric acid and linoleic acid nanoparticles prepared with double-surfactant system; 5-FU, 5-fluorouracil.

Figure 4

FTIR spectra of FLOM, FLOD and FLLD. Abbreviations: FTIR, Fourier transform infrared spectroscopy; FLOM, 5-FU-loaded lauric acid and oleic acid nanoparticles prepared with mono-surfactant system; FLOD, 5-FU-loaded lauric acid and oleic acid nanoparticles prepared with double-surfactant system; FLLD, 5-FU-loaded lauric acid and linoleic acid nanoparticles prepared with double-surfactant system; 5-FU, 5-fluorouracil.

Figure 5

Graphical presentation of the (A) calibration curve of 5-FU prepared by using UV–Vis spectrophotometer and (B) 5-FU release from SLNs at 37°C (solid lines) and 39°C (dashed lines) by using USP dissolution apparatus (data = mean ± SD, n=3). Abbreviations: 5-FU, 5-fluorouracil; UV-Vis, ultraviolet-visible; SLN, solid lipid nanoparticle; FLOM, 5-FU-loaded lauric acid and oleic acid nanoparticles prepared with mono-surfactant system; FLOD, 5-FU-loaded lauric acid and oleic acid nanoparticles prepared with double-surfactant system; FLLD, 5-FU-loaded lauric acid and linoleic acid nanoparticles prepared with double-surfactant system; FOD, 5-FU-loaded oleic acid microemulsion with double-surfactant system; FOM, 5-FU-loaded oleic acid microemulsion with mono-surfactant system; USP, United States Pharmacopeia.

Figure 6

DPV method for the (A) DPV response to different concentrations of 5-FU, (B) calibration curve of 5-FU as plot of drug concentration and measured electric current, and (C) 5-FU release from SLNs at 37°C (solid lines) and 39°C (dashed lines) (data = mean ± SD, n=3). Abbreviations: DPV, differential pulse voltammetry; 5-FU, 5-fluorouracil; SLN, solid lipid nanoparticle; FLOM, 5-FU-loaded lauric acid and oleic acid nanoparticles prepared with mono-surfactant system; FLOD, 5-FU-loaded lauric acid and oleic acid nanoparticles prepared with double-surfactant system; FLLD, 5-FU-loaded lauric acid and linoleic acid nanoparticles prepared with double-surfactant system.

Figure 7

Biocompatibility and cytotoxicity of the (A) blank and 5-FU-loaded SLNs against HGF cells after 24 hours and (B) 5-FU-loaded SLNs against breast cancer cells (MDA-MB-231) after 1 hour at 37°C and 39°C (data = mean ± SD, n=3). Note: (B) 37 and 39 represent temperature (°C) of drug release studies. Abbreviations: 5-FU, 5-fluorouracil; SLN, solid lipid nanoparticle; HGF, human gingival fibroblast; FLOM, 5-FU-loaded lauric acid and oleic acid nanoparticles prepared with mono-surfactant system; FLOD, 5-FU-loaded lauric acid and oleic acid nanoparticles prepared with double-surfactant system; FLLD, 5-FU-loaded lauric acid and linoleic acid nanoparticles prepared with double-surfactant system; LOM, lauric acid and oleic acid nanoparticles prepared with mono-surfactant system; LOD, lauric acid and oleic acid nanoparticles prepared with double-surfactant system; LLD, lauric acid and linoleic acid nanoparticles prepared with double-surfactant system.