(M1130-11-63) Synthesis of Novel Surfactant for Formulating Cationic Nanoparticles

Monday, October 21, 2024

11:30 AM – 12:30 PM MT

Presenting Author(s)

BR

Bernadette Riemann

PhD student
University of Bonn
Bonn, Nordrhein-Westfalen, Germany

Main Author(s)

BR

Bernadette Riemann

PhD student
University of Bonn
Bonn, Nordrhein-Westfalen, Germany

Co-Author(s)

JS

Jakob Schiwy

PhD Student
University of Bonn
Bonn, Nordrhein-Westfalen, Germany
Alf Lamprecht

Professor
University of Bonn
Bonn, Nordrhein-Westfalen, Germany

Purpose: In recent studies, polymeric nanoparticles (NP) were often deployed to deliver immunomodulating drugs to the inflammation side in the body in aim to reduce unwanted side effects. Here, the polymer poly-(DL)-lactide-co-glycolide (PLGA) is often utilized as a biocompatible material for the production of NPs. It is generally stabilized with surface active polymers (e.g. poly vinyl alcohol or polysorbate) producing non-ionic NPs. However, little attention has been paid to low molecular emulsifiers as stabilizers for nanoparticles. Individual studies showed that PLGA NPs can also be formed by using didodecyldimethylammoniumbromid (DMAB) as a cationic surfactant [1]. However, the full potential of low molecular ionic surfactants as equivalent stabilizers for NP formulation has not yet been fully explored. In this study, we aimed to develop a new cationic surfactant (LAN⁺) for the stabilization of PLGA NPs. The surfactant is based on linolenic acid, an essential unsaturated fatty acid, which is a major contributor in the formation of various anti-inflammatory prostaglandins. By combining NPs with stabilizers based on anti-inflammatory fatty acids, we seek to further enhance the efficiency of inflammatory disease drug therapy.

Methods: The cationic surfactant was synthesized by mixing 1.1 equivalents of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) with 1.1 equivalents of the fatty acid (linolenic acid) and 4 equivalents of N, N-diisopropylethylamine (DIPEA) in dry dimethylformamide (DMF) and further adding the amine ((2-aminoethyl)trimethylammoniumchloride hydrochloride) after 15 minutes (figure 1). The reaction mixture was kept at room temperature (20 ± 2 °C) for 16 hours, diluted with water afterwards and filtrated through a 0.2 µm PTFE filter to remove undissolved side products. Subsequently, the aqueous solution was purified by solid phase extraction using a weak cation exchanger column (Strata™-XL-CW 100µm polymeric weak cation column purchased by Phenomenex®, US). Therefore, the solid phase was conditioned with methanol, equilibrated with water, loaded with the aqueous sample washed with water, methanol and acetonitrile and finally eluted with methanolic formic acid (16.67% v/v). The identity of the final product LAN⁺, a pale-yellow liquid, was confirmed by LCMS measurement. The product of the synthesis (LAN⁺) was then utilized as a stabilizer for PLGA NPs. The particles were prepared based on the oil-in-water (O/W) emulsion solvent evaporation technique described by Ries et al. [2]. Briefly, 10 mg of PLGA (PURASORB® PDLG 500 2A from Corbion, NL) were dissolved in ethyl acetate and then slowly added to the aqueous solution of the stabilizer during sonication for five minutes. After evaporating the organic phase with a rotation evaporator, the particle size and zeta potential of the formulated nanoparticles (PLGA-LAN-NP) were measured (HORIBA Scientific nanoPartica SZ-100, Japan). Additionally, the stability of the dispersions stored at 4 ± 2 °C was determined and electron microscopic images were taken by Helios G4 CX Dualbeam microscope (Thermo Fisher Scientific, NL) after washing and freeze-drying of the PLGA-LAN-NP dispersion in an Alpha 1-4 LSC Plus freeze-dryer (Martin Christ, G).

Results: The LCMS results confirmed the successful amide coupling and purification (363.34 m/z). The novel cationic surfactant furthermore proved to stabilize the formulated PLGA-LAN-NPs in a wide range of concentrations (0.48-3.44 mg/ml). Particle size (z average) ranged between 45.5 ± 1.6 nm and 124.0 ± 3.3 nm, the zeta potential for all PLGA-LAN-NPs was 52.23 mV to 73.22 mV, respectively. In comparison, PLGA NPs stabilized with polyvinyl alcohol (1%), prepared with the same method, showed a slightly negative zeta potential (-2.57 ± 1.03mV), indicating more stable formulations by depolying the novel surfactant. The particle size of the PLGA-LAN-NPs increased with decreasing concentration of LAN⁺, while maintaining a narrow size distribution as shown in figure 2A. Furthermore, the stability of the novel cationic PLGA-LAN-NPs was examined. All formulations containing the novel surfactant LAN⁺ showed no significant change (p < 0.05) of the particle size during three days of storage at 4 ± 2 °C (figure 2B). In addition, the particle size was confirmed via electron microscopy and also showed the spherical morphology of the formulated PLGA-LAN-NPs (figure 3).

Conclusion: The novel cationic surfactant was successfully synthesized and deployed as an effective stabilizer for the formulation of PLGA NPs. The size of the particles directly depended on the concentration of the novel stabilizer, enabling the purposeful design of NPs in a certain size range. No change in particle size was detected in the course of at least three days at 4 °C. The presented results suggest exciting new possibilities for the formulation of biocompatible NPs based on small cationic emulsifiers. Prolonged stability studies, as well as toxicity and immunological effect studies on different cells and in inflamed tissues can be conducted to gain a deeper insight into the mechanisms governing stabilization and effectiveness of small molecular charged surfactants and NPs.

References: [1] Bhardwaj V, Ankola DD, Gupta SC et al. (2009) PLGA nanoparticles stabilized with cationic surfactant: safety studies and application in oral delivery of paclitaxel to treat chemical-induced breast cancer in rat. Pharm Res 26:2495–2503.
[2] Ries M, Moulari B, Shetab Boushehri MA et al. (2022) Adalimumab Decorated Nanoparticles Enhance Antibody Stability and Therapeutic Outcome in Epithelial Colitis Targeting. Pharmaceutics 14.

Acknowledgements: The authors thank the Cusanuswerk for a PhD scholarship for B.R.

Amid coupling reaction schema of linolenic acid (A) with (2-aminoethyl)trimethylammoniumchloride (B) to the cationic surfactant LAN+ (C); reaction conditions(1): dry DMF, 20 ± 2°C, 16h, 1.1 eq. HATU, 4 eq. DIPEA

Particle size distribution of the PLGA NPs with different concentrations of the novel stabilizer LAN+ (A), and the stability of the PLGA NP formulations for three days stored at 4 ± 2 °C (B).

(A-D) Electron microscopy images of the PLGA-NP formulated with cationic surfactant (1.43mg/mL stabilizer LAN+).