(T1030-07-45) NMR Analysis of Molecular Interactions of Polyoxyethylated Amphiphiles for Solid Micellar-Size Nanoparticles: Probing into the Mechanism of Anti-Dilution Properties

Purpose: In drug development and delivery, poor aqueous solubility remains a major barrier for many therapeutic compounds. Micellar solubilization is a commonly employed strategy to address this challenge; however, traditional micellar systems are prone to dissociation upon dilution, limiting their effectiveness for solubilizing poorly water-soluble drugs. Solid micellar-size nanoparticles (SMNP) formulated using nonionic polyoxyethylated (POE) amphiphiles such as Brij®76 have demonstrated improved solubility, stability, and resistance to dilution^2,3, primarily due to the temperature-dependent changes in the physical state of the nanoparticles during formulation. Nonionic POEs, similar to Brij®76, have also been shown to exhibit temperature-sensitive phase transitions within the hydrophobic blocks that form the micellar core⁴. This project aims to investigate the temperature-dependent behavior of polyoxyethylated amphiphiles used in SMNPs at a molecular level to better understand their contribution to the enhanced stability and anti-dilution behavior of these systems.

Methods: Solid micellar-size nanoparticles consisting of only POE (10) stearyl ether (Brij®76) were prepared by forming a solution above the primary surfactant’s Krafft point 45°C and quenching in an ice bath to 0°C. The critical micelle concentration (CMC) was determined through pyrene quenching using the RF-5301 spectrofluorophotometer (Shimadzu, JPN). All NMR experiments were conducted on a JEOL-ECA-600 (JEOL, USA)operating at a 1H frequency of 600 MHz at 25°C with normal geometry and z-axis gradient shims, in 5 mm OD NMR tubes with a coaxial insert of 100 mL D₂O. All experiments were done at temperatures between 3°C to 50°C with spinning (15 Hz). All spectra are referenced to water at δ 4.67 ppm and Fourier-transformed, processed, and overlaid with Delta software (v.6.1.0).m

Results: Figure 1 presents the ¹H NMR of Brij®76 SMNP solutions, with peak assignments [a] to [f], excluding the water signal at 4.67 ppm. Clear temperature-dependent changes were observed in the spectra (Figure 2), reflecting a shift in molecular interactions with cooling. At lower temperatures, peaks associated with the hydrocarbon chains in the core ([a]-[c]) exhibited decreased peak intensity and broadening, indicating a substantial reduction in the segmental mobility of the encapsulated hydrocarbon chains, consistent with increased rigidity within the micellar core. This points toward a phase behavior shift towards a more rigid architecture during the cooling process. Additionally, a downfield chemical shift from 0.53365 ppm at 15°C to 0.54297 ppm at 3°C  in the innermost CH3 group [a] suggests a temperature-induced conformational rearrangement, likely reflecting a more ordered core arrangement under low temperature conditions (Figure 2). Further temperature-dependent analysis of the 22 mM Brij®76 SMNP formulation shows a sharp decline in peak intensity for peaks [b] and [c] between the temperatures of 45°C and 50°C, indicating a disruption in the SMNP’s molecular arrangement (Figure 3). This coincides with the known Krafft point of 45°C for Brij®76, where at temperatures above this threshold, the surfactant transitions from a solid to liquid phase, resulting in a breakdown of the structured assembly induced by quenching. These NMR findings are further corroborated by critical micelle concentration measurements, which reflect the SMNP system’s stability against dilution. The CMC for non-quenched Brij®76 only samples was determined to be 0.0046 mM, 3-fold higher than the previously reported literature value of 0.00156 mM¹. In contrast, quenched Brij®76 SMNP samples showcased a significantly lower CMC value of 0.00035 mM, over 13-fold less than that of their non-quenched counterparts and closely aligning with previously determined apparent CMC value of 0.0004 mM² (Figure 4).

Conclusion: This study highlights the temperature-dependent molecular interactions of quenched Brij®76 SMNPs, which contribute to their enhanced structural integrity and anti-dilution behavior. The ¹H NMR spectra indicate that at lower temperatures, such as those during the quenching process, the micellar core adopts a more rigid and ordered conformation, likely due to reduced hydrocarbon chain mobility. Notably, additional spectral changes near the Krafft point (45°C) suggest a distinct phase transition, underscoring the influence of temperature on the structural organization of the SMNPs. The enhanced structural stability was further supported by critical micelle concentration measurements, which confirmed that SMNPs exhibited stronger resistance to dilution. Collectively, these findings underscore the enhanced stability and anti-dilution capabilities of Brij®76-based SMNP systems, emphasizing their potential for effective delivery of poorly water-soluble drugs.

References: 1. Gao, Xiaoling et al. “Design and preparation of nanostructures based on Krafft point of nonionic amphiphiles for delivery of poorly water-soluble compounds.” International journal of pharmaceutics vol. 588 (2020): 119789. doi:10.1016/j.ijpharm.2020.119789
2. Li, J (2024, July 8-12) Thermodynamic Insights into the Enhanced Stability and Anti-dilution Properties of Solid Micellar-size Nanoparticles [Poster presentation]. Controlled Release Society, Bologna, Italy
3. Li, J (2024, October 20-23) Role of Temperature in the Formation of Solid Micellar-size Nanoparticles of Nonionic Polyoxyethylated Amphiphiles [Poster Presentation]. AAPS PharmSci 360, Salt Lake City, UT.
4. Plazzotta, B., Dai, J., Behrens, M. A., Furó, I., & Pedersen, J. S. “Core Freezing and Size Segregation in Surfactant Core–Shell Micelles” The Journal of Physical Chemistry B, vol.119 (2015), 10798–10806. doi:10.1021/acs.jpcb.5b06041


Figure 3: 1H NMR spectra of 22 mM Brij®76 SMNP formulation at different temperatures between 25°C and 50°C.

Figure 4: At 25°C, non-quenched Brij®76 solutions (A) had a CMC of 0.0046 mM, nearly 3-fold higher than previously determined CMC of 0.00156 mM. Quenched solutions (B) had a CMC of 0.00035 mM, over 13-fold lower than non-quenched samples.


Figure 1: The 1H NMR spectrum of primary surfactant Brij®76 only SMNP solution in coaxial insert with D2O, with peaks of interest labeled corresponding to the molecular structure of the molecule. The water peak of 4.67 ppm is not shown.

Figure 2: 1H NMR spectra of 22 mM Brij®76 formulation at different temperatures between 3°C and 15°C, with zoomed in insets corresponding to peak [b] and [a] at higher resolution.