A quality by design approach for the synthesis of palmitoyl-L-carnitine-loaded nanoemulsions as drug delivery systems

Abstract

Nanoemulsions (NE) are lipid nanocarriers that can efficiently load hydrophobic active compounds, like palmitoyl-L-carnitine (pC), used here as model molecule. The use of design of experiments (DoE) approach is a useful tool to develop NEs with optimized properties, requiring less experiments compared to trial-and-error approach. In this work, NE were prepared by the solvent injection technique and DoE using a two-level fractional factorial design (FFD) as model was implemented for designing pC-loaded NE. NEs were fully characterized by a combination of techniques, studying its stability, scalability, pC entrapment and loading capacity and biodistribution, which was studied ex-vivo after injection of fluorescent NEs in mice. We selected the optimal composition for NE, named pC-NE_U, after analysis of four variables using DoE. pC-NE_U incorporated pC in a very efficient manner, with high entrapment efficiency (EE) and loading capacity. pC-NE_U did not change its initial colloidal properties stored at 4 °C in water during 120 days, nor in buffers with different pH values (5.3 and 7.4) during 30 days. Moreover, the scalability process did not affect NE properties and stability profile. Finally, biodistribution study showed that pC-NE_U formulation was predominantly concentrated in the liver, with minimal accumulation in spleen, stomach, and kidneys.

Graphical Abstract

Keywords:

• Nanoemulsion
• drug delivery
• design of experiments
• colloidal stability
• biodistribution

Acknowledgments

We thank Laura Barrios from Secretaría General Adjunta de Informática (SGAI, CSIC) (Spain) for her help and suggestions for interpretation and discussion of DoE results, and Paolo Gasco from Nanovector Srl. (Italy) for his advices during chemical characterization of pC-NE_U, performed in Nanovector’s headquarters.

Ethical approval statement

Mice were housed in the specific pathogen-free facilities at the Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid (Spain). All animal experiments conformed to EU Directive 2010/63EU and Recommendation 2007/526/EC, enforced in Spanish law under Real Decreto 53/2013. The protocol was approved by the Madrid regional government (PROEX277/16).

Mice of mean weight 25 g were housed (<5 per cage) with food and water ad libitum, in ventilated controlled racks (T 20-24 °C, 45–65% humidity, 12/12 h light/dark cycle). Mechanical restraint was always prioritized versus sedation.

Authorship contribution statement

Eva Maria Arroyo-Urea: Investigation, Data curation, Writing – original draft preparation, Visualization. María Muñoz-Hernando: Methodology, Investigation, Visualization. Marta Leo-Barriga: Investigation, Visualization. Fernando Herranz: Conceptualization, Methodology, Supervision, Visualization, Writing – review & Editing, Funding acquisition. Ana González-Paredes: Conceptualization, Formal analysis, Supervision, Visualization, Writing – review & editing, Funding acquisition.

Disclosure statement

The authors report there are no competing interests to declare.

Additional information

Funding

This work was supported by Atracción de Talento (Modalidad 1) program from Comunidad de Madrid (Spain) (Reference 2019-T1/IND-12906) and Spanish Ministry of Science (Grants nos. PID2019-104059RB-I00 and PDC2022-133493-I00).