Focus on the importance of the process parameters: Temperature cycles and “shock” water addition during the phase inversion process for the formulation of lipid nanoparticles

Abstract

The phase inversion temperature method is one of the formulation processes to elaborate nanomedicine based on lipid nanoparticles. This process was used in numerous studies in literature, changing the nature of the ingredients to ensure good encapsulation efficiencies of various drugs. While the product quality attributes to develop various nanomedicines were largely explored, the process parameters remained unchanged from the first lipid nanoparticles designed with this process 25 years ago. It is always composed of 3 temperature cycles and a fast addition of cold water, creating an “irreversible shock” to obtain the lipid nanoparticle in suspensions. To date, the exact roles of these 2 steps remain unclear. We decided to explore their impact by changing the number of temperature cycles and by modifying the final addition of water (temperature, salinity, with or without). Using these various conditions, the size distribution and stability, the capacity of encapsulation and the cytotoxic property of the lipid nanoparticles were compared. It demonstrated the indispensableness of these parameters: at least 1 temperature cycle and rapid cooling and dilution of the mixture, to obtain the most performant nano-systems.

Introduction

Lipid nanoparticles (LNP), one of the nanovectors used for many years in attempts to develop nanomedicine, have seen renewed interest with the COVID-19 pandemic and the successful clinical development of mRNA vaccines [1]. Numerous manufacturing processes are reported in the literature, both batch processes such as the self-nanoemulsifying drug delivery systems [2], and continuous processes using microfluidic systems [3].

One of the batch formulation processes used to develop LNP is based on the phase-inversion temperature (PIT) method first described in the late sixties by Shinoda et al. [4]. Heurtault et al. adapted this concept to develop LNP based on an oily core surrounded by a mixed layer of lecithins and a pegylated surfactant, and patented the process [5], [6], [7]. Basically, under magnetic stirring, three cycles of progressive heating and cooling are performed, between 65 and 95°C, on the mixture of three main components: oil (initially Labrafac® WL1349), non-ionic surfactants (initially Lipoid® S75–3 and Kolliphor® HS-15), and salted water. High conductivity values were obtained at low temperature corresponding to an aqueous continuous phase: an oil-in-water (o/w) emulsion while at high temperature, low conductivity values (close to 0 mS/cm) were obtained meaning that the continuous phase is the oil, so a water-in-oil (w/o) emulsion. This phase inversion was defined in a temperature range, a phase inversion zone (PIZ), corresponding to a micro-emulsion state, where no dispersed and continuous phase can be observed [5]. During the last cooling, in the PIZ (about 75°C using the initial components), a rapid addition of cold (4°C) pure water is operated. In the patent and the first publications regarding the LNP formulation process, the role the water addition is confused. The authors defined the operation as a dilution process that breaks off the microemulsion state, leading to an irreversible shock and the LNP formation [5], [6], [7]. Depending on the proportion of oil and non-ionic surfactants, the LNP size can be controlled (hydrodynamic diameter range from 25 to 100 nm), with a very narrow size distribution. These LNP characteristics are very well controlled with this solvent-free, soft-energy method, and a good stability profile in an aqueous environment for up to 18 months was maintained [5], [6], [7].

Over the past two decades, the LNP were extensively used as nanomedicines for various pre-clinical applications: cancer (liver, lung, brain), inner ear therapies, drug oral administration, etc. The surfactant monolayer structure and the oily core of LNP allowed amphiphilic and lipophilic drugs, respectively, to be easily incorporated inside the nanocarriers. So a large variety of drugs have been loaded in LNP such as amiodarone [8], ibuprofen [9], indinavir [10], etoposide [11], paclitaxel [12], [13], [14], [15], [16], tripentone [17], Sn38 [18], Rhenium complex [19], [20], derivatives of 4-hydroxy tamoxifen combined with ferrocene [21], [22], [23], [24], cannabidiol [25], albendazole [26], amphiphilic-modified 5-FU [27], amphiphilic modified gemcitabine [28], [29], [30]. Moreover, iron oxide [31], fluorinated molecules [32], or electron paramagnetic resonance probes [33], were encapsulated in LNP to visualize their distribution or assess oxygenation after in vivo administration. Finally, biological systems were loaded or adsorbed in the core or at the surface, respectively, of LNP such as targeting peptides [34], [35], antimicrobial peptides [36], [37], DNA [38], [39], and siRNA [40], [41], [42], [43].

For this purpose, the formulation was modified in terms of components to perform these drug-loaded LNP depending on the drug properties such as the solubility in the oil core and the capacity to be adsorbed at the LNP surface. For example, Labrafac® WL1349 was replaced by Captex® 8000 to ensure a better paclitaxel or albendazole encapsulation rate [12], [13], [14], [15], [16], [26], or by Ethyl Oleate® leading to higher loading of iron oxide [31]. Using the classical components, Cholesterol, Span® 80, glycerol monolaurate or a mixture of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) / 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) was added to improve the encapsulation rate of idinavir [10], amphiphilic modified gemcitabine and 5-FU [27], [28], [29], [30], antimicrobial peptides [36], [37], or nucleic acid materials [24], [38], [39], [42]. Complexes of DNA were encapsulated in LNCs using an additional surfactant: oleic plurol® with Kolliphor® HS-15 and permitted an incorporation of DNA complex inside LNP at moderate temperature, without affecting the biological effect [38], [39]. In addition, Béduneau et al. replaced Kolliphor® HS-15 with DUB SPEG 30S®: a surfactant with a longer PEG chain, to improve their furtivity in the systemic circulation [44]. To load Sn38, Roger et al. totally modified the formulation components with the use of Transcutol® HP as surfactant and Labrafil® M 1944 CS as oil since the drug is not soluble in Labrafac® WL1349 [18].
The only common features of this non-exhaustive list of drug-loaded LNP are the formulation steps when using the phase inversion process. Whatever the component modifications, the protocol always consisted of temperature cycles (usually 3 cycles, although some studies have modified this number [45]) around the microemulsion state, followed by the irreversible shock by the rapid dilution with an aqueous phase (containing additives or not) at the ZIP temperature usually.

In this study, the role and the importance of the number of temperature cycles as well as the rapid cold dilution with pure water were shown and a simplified PIT method was defined. Using the standard compounds: Labrafac® WL1349, Lipoid® S75–3 and Kolliphor® HS-15, four LNP sizes with hydrodynamic diameters of 25, 50, 75 and 100 nm were studied. The various batches of LNP performed with classic and simplified formulation protocols were compared in terms of size distribution, stability, capacity for the encapsulation of model molecules, cytotoxic behavior and the presence of nano-impurities (such as surfactant micelles). All these experiments led to new advances in the comprehension about the phase inversion temperature method and the importance of the irreversible shock.

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Materials

The amounts of Labrafac® WL 1349 (Lab) (triglycerides of caprylic and capric acids) (Gattefossé S.A., Saint-Priest, France) (oil phase); water (Milli-Q plus® system, Millipore, Billerica, MA, USA) and NaCl (Sigma–Aldrich, Saint-Quentin-Fallavier, France) (aqueous phase); and Kolliphor® HS-15 (Kol) (mixture of free polyethylene glycol 660 and polyethylene glycol 660 hydroxystearate) (BASF, Ludwigshafen, Germany) and Lipoïd® S75–3 (Lip) (soybean lecithin: mainly phosphatidylcholine (69 %).

Claire Gazaille, Sonia Pîgleşan, Kristóf Apró, Laura Ruesche, Aouatef Foudi, Adélie Mellinger, Benjamin Siegler, Joël Eyer, Laurent Lemaire, Patrick Saulnier, Florence Franconi, Guillaume Bastiat, Focus on the importance of the process parameters: Temperature cycles and “shock” water addition during the phase inversion process for the formulation of lipid nanoparticles, Colloids and Surfaces A: Physicochemical and Engineering Aspects, Volume 712, 2025, 136428, ISSN 0927-7757, https://doi.org/10.1016/j.colsurfa.2025.136428.