(W1030-09-59) Investigation of Advanced Fattigation-Platform Using PLGA-Cystamine-Fatty Acid Conjugates for Effective Tucatinib Delivery

Purpose: The objective of this study was to develop a novel PLGA nanoparticle (NP) system for subcutaneous delivery of tucatinib. Tucatinib is currently used for the treatment of HER2-overexpressing breast cancer. It is administered via oral route with frequent dosing requirements. The reference drug product of tucatinib requires 150 mg twice daily dosing for HER2-overexpressing breast cancer, leading to compliance challenges and fluctuating plasma levels, necessitating a long-acting delivery system. To address this limitation, PLGA was conjugated with cystamine and fatty acids of varying chain lengths, specifically decanoic acid (C10) and stearic acid (C18), through 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide (EDC/NHS) coupling reactions. The investigation focused on evaluating how cystamine conjugation and fatty acid chain length influence nanoparticle properties, drug encapsulation, and release behaviors to establish optimal formulation parameters for improved patient compliance.

Methods: PLGA-CYS conjugates were synthesized via EDC/NHS-mediated coupling between PLGA (41.4 mg, 3 μmol) dissolved in dichloromethane and cystamine dihydrochloride (1.35 mg, 6 μmol), followed by purification through diethyl ether precipitation to remove unreacted materials and centrifugation. PLGA-CYS-fatty acid conjugates were prepared by reacting PLGA-CYS with fatty acids (decanoic acid: 1.03 mg, stearic acid: 1.71 mg, 6 μmol) using EDC/NHS reagents. The final synthesized products were dried in a fume hood for 18 hours. Conjugation was confirmed by ¹H-NMR analysis. NPs were formulated using an O/W emulsion method. The organic phase containing polymer (PLGA, PLGA-CYS, or PLGA-CYS-fatty acid) and tucatinib (2 mg), a poorly water-soluble drug, dissolved in DCM was added dropwise into 8 mL of 1% polyvinyl alcohol solution (9000-10000 Da), followed by probe sonication, solvent evaporation, and ultracentrifugation. Purified NPs were freeze-dried for storage. Particle size distribution, zeta potential, and morphologies were characterized using dynamic light scattering (DLS) and field emission scanning electron microscopy (FE-SEM). Drug loading content was determined by dissolving freeze-dried NPs in solvent followed by reverse-phase HPLC (RP-HPLC) analysis. Encapsulation efficiency was calculated using the supernatant obtained during purification via HPLC analysis. In vitro release profiles were assessed in phosphate-buffered saline at different pH conditions (pH 5.5 for cancer cells, pH 7.5 for normal cells) under physiological conditions for up to 72 hours to evaluate long-acting performance under bioequivalent conditions.

Results: Four nanoparticle formulations were prepared: PLGA NPs, PLGA-CYS NPs, PLGA-CYS-decanoic acid (C10) NPs, and PLGA-CYS-stearic acid (C18) NPs. ¹H-NMR analysis confirmed conjugation, showing characteristic PLGA peaks at 5.2 ppm (methine proton of lactic acid), 4.8 ppm (methylene proton of glycolic acid), and 1.5 ppm (methyl proton of lactic acid) in all samples (Figure 1). Cystamine conjugation was evidenced by new peaks at 2.8-3.2 ppm corresponding to -CH₂-CH₂-NH- and -CH₂-CH₂-S-S- protons. Fatty acid conjugation was confirmed by characteristic aliphatic chain signals: (CH₂)n peaks at 1.2-1.4 ppm and terminal CH₃ peaks at 0.8 ppm. All NPs exhibited sizes between 200-300 nm by DLS analysis, with PLGA-CYS-C18 NPs showing the smallest size (203.8 ± 5.8 nm) and PLGA-CYS-C10 NPs the largest (287.2 ± 33.1 nm) (Figure 2). Zeta potential values were approximately -30 mV for all formulations, indicating negatively charged surfaces. FE-SEM revealed spherical morphology with modified surface topology in fatty acid-conjugated formulations compared to smooth PLGA NPs. Drug loading and encapsulation efficiency varied among formulations (Figure 3). PLGA NPs achieved the highest drug loading content (6.13 ± 0.10%) with encapsulation efficiency of 65.45 ± 0.15%. Cystamine conjugation reduced drug loading to 1.60 ± 0.02% while improving encapsulation efficiency to 74.64 ± 0.17%. Fatty acid conjugation further enhanced encapsulation performance: PLGA-CYS-C10 NPs showed 1.04 ± 0.02% drug loading with 75.11 ± 0.94% efficiency, while PLGA-CYS-C18 NPs demonstrated 0.71 ± 0.04% loading with the highest encapsulation efficiency (81.76 ± 0.29%). This trend suggests that longer fatty acid chains provide better hydrophobic interactions with tucatinib. In vitro release profiles at different pH conditions (pH 5.5, 7.5) revealed that fatty acid conjugation significantly modified drug release patterns compared to conventional PLGA NPs. PLGA NPs exhibited typical burst release characteristics across all pH conditions, while PLGA-CYS-fatty acid NPs achieved more sustained release profiles with reduced initial drug release. All formulations showed enhanced drug release under acidic conditions (pH 5.5) compared to physiological pH (7.5), demonstrating pH-responsive release behavior potentially beneficial for tumor microenvironment targeting.

Conclusion: PLGA-cystamine-C18 conjugate NPs demonstrated favorable physicochemical characteristics among tested formulations, achieving highest encapsulation efficiency (81.76%) and smallest particle size (203.8 nm). Modification of PLGA with cystamine and fatty acids altered NP properties, with C18 derivatives showing superior drug encapsulation compared to conventional PLGA NPs (65.45% efficiency). Fatty acid chain length modulation reduced burst release and extended drug release kinetics over 72 hours. pH-responsive release behavior across formulations indicates applicability for targeted delivery. This study demonstrates that cystamine-fatty acid conjugation can enhance PLGA-based nanoparticle performance for tucatinib encapsulation and provides foundation for developing sustained-release formulations that may reduce current twice-daily dosing requirement.

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Acknowledgements: This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education (RS-202300245029).

Figure 1: ¹H-NMR spectra confirming successful conjugation of PLGA with cystamine and fatty acids

Figure 2: Particle size distribution, zeta potential, and FE-SEM images of formulated nanoparticles

Figure 3: Comparative analysis of drug loading contents and encapsulation efficiency