NOVEL ENDOSOMAL ESCAPE ENHANCERS ENABLE EFFICIENT INTRACELLULAR DELIVERY OF THERAPEUTIC NANOPARTICLES TO BREAST CANCER CELLS DISSERTATION Written work as one of the requirements to obtain a Doctoral degree from Institut Teknologi Bandung By: EBRAHIM SAAD EBRAHIM SADAQA NIM: 30722701 (Program Study Doctoral of Pharmacy) INSTITUT TEKNOLOGI BANDUNG March 2025 i ABSTRACT NOVEL ENDOSOMAL ESCAPE ENHANCERS ENABLE EFFICIENT INTRACELLULAR DELIVERY OF THERAPEUTIC NANOPARTICLES TO BREAST CANCER CELLS By Ebrahim Saad Ebrahim Sadaqa STUDENT ID: 30722701 (Doctoral Program in Pharmacy) Endosomal entrapment remains a major challenge in the intracellular delivery of therapeutic nanoparticles, leading to lysosomal degradation and reduced therapeutic efficacy. Following endocytosis, many nanoparticles remain sequestered in endosomes and aresubsequently trafficked to lysosomal degradation pathways, preventing cytoplasmic drug release. Various endosomal escape mechanisms, including membrane fusion, pore formation, and membrane disruption, have been explored to enhance nanoparticle delivery. The aim of this study was to develop two novel endosomal escape strategies: sodium oleate (NaOL) as a lipid-based membrane fusion agent and the rationally designed peptide H3L5E6D10H12for controlling endosomal pore formation. By leveraging these mechanisms, this research sought to enhance the intracellular delivery efficiency of therapeutic nanoparticles to triple- negative breast cancer (TNBC) cells. For the first approach,in silicostudy by molecular dynamics (MD) simulations were employed to identify and assess candidate endosomal fusion agents, focusing on lipid- membrane interactions and their effects on membrane structure. The validation was experimentally conducted by modifyingsimvastatin-loaded liposomes with sodium oleate using the thin-film hydration method, followed by comprehensive characterization of particle size, polydispersity index (PDI), zeta potential, and drug encapsulation efficiency analysis. Additionally,in vitroassessments were performed to evaluate the anticancer effects of the modified liposomes on TNBC 4T1 cells, including subcellular colocalization analysis, cellular uptake studies, reactive oxygen species (ROS) production, DNA damage quantification, and apoptosis induction. The peptide H3L5E6D10H12was rationally designed using anin silicoapproach inspired by the antimicrobial peptide Temporin L. MD simulations (100 ns) were conducted to optimize its stable conformation,membrane interactions, and structural adaptability under endosomal conditions. The peptide was engineered to remain neutral at physiological pH while progressively acquiring a positive charge in the acidic endosomal environment (pH≈5), enhancing interactions with negatively charged phospholipid head groups and facilitating controlled pore formation.Followingin silicooptimization, the peptide was experimentally conjugated to liposomes and endosomal escape efficiency was then assessed through subcellularcolocalization analysis. ii MD simulations for 200 ns revealed that NaOL undergoes protonation in the acidic endosomal environment (pH≈5), enhancing hydrophobic interactions with the membrane and inducing structural disruption that facilitates liposomal cargo release into the cytoplasm. NaOL promotes endosomal membrane fusion by increasing lipid tilt, decreasing membrane thickness, and inducing negative membrane curvature which are the key factors in promoting vesicle destabilization and fusion events. Dynamic light scattering (DLS)analysis revealed that unmodified liposomes had an average diameter of 115.2 ± 7.9 nm, whereas sodium oleatemodified liposomes (Sim- NaOL-Lipo) maintained a particle size of 119 ± 9.37 nm, maintaining a stable polydispersity index (PDI) of 0.206 ± 0.011, indicative of a monodisperse population. Zeta potential measurements showed a shift from-9.67 ± 3.01 mV in unmodified liposomes to-31.05 ± 2.38 mV after NaOL modification, reflecting changes in surface charge that improve colloidal stability.In vitrostudies using TNBC 4T1 cells demonstrated that sodium oleate-modified liposomes facilitated efficient endosomal escape, as evidenced by reduced lysosomal colocalization observed in confocal microscopy analysis at both 1 and 3 hours postincubation. Sim-NaOL-Lipo promoted membrane fusion and the therapeutic release of simvastatin, which led to significantly elevated ROS production, as confirmed by increased DCF fluorescence intensity. Specifically, Sim-NaOL-Lipo induced a significant increase in ROS levels (p < 0.001) compared to unmodified Sim-Lipo. The unmodified Sim-Lipo failed to provoke significant ROS activity beyond baseline levels (p = 0.475), underscoring the critical role of surface modifications in enhancing intracellular oxidative stress. The elevated ROS levels induced by Sim-NaOL-Lipo correlated with enhanced DNA damage (p < 0.001) and apoptosis, culminating in superior cytotoxic efficacy. Cytotoxicity assays revealed a significant reduction in IC50values for Sim-NaOL-Lipo (9.1 ± 4.58 µg/mL) comparedto Sim-Lipo (26.9 ± 8.05 µg/mL), representing a 2.9-fold improvement. These findings highlight the ability of sodium oleate modifications to enhance intracellular delivery and therapeutic outcomes by facilitating endosomal escape and promoting oxidative stress. The peptide H3L5E6D10H12was designed to facilitate controlled endosomal pore formation. MD simulations (100 ns) demonstrated an average number of 6.21 ± 2.98 hydrogen bonds with phospholipid head groups, coupled with a reduction in membrane thickness from 39.23 ± 0.43 Å to 39.08 ± 0.55 Å, indicative of strong peptide-membrane interactions. Experimental evaluation of peptide-conjugated liposomes confirmed enhanced endosomal escape through reduced lysosomal colocalization, validating the computationalpredictions. These findings establish sodium oleate and H3L5E6D10H12peptide as two distinct yet complementary endosomal escape enhancers. Sodium oleate facilitates endosomal fusion and enhances simvastatin therapeutic release, while the peptidebased strategy promotes pH-triggered pore formation for controlled endosomal escape. This study provides new insights into designing more efficient nanoparticlebased drug delivery systems, with potential applications in cancer therapy and other diseases requiring precise intracellular drug distribution. Keywords: Endosomal escape, liposomes, simvastatin, sodium oleate, H3L5E6D10H12 peptide, Triple negative breast cancer. iii ABSTRAK PENGUAT PELEPASAN ENDOSOMAL BARU UNTUK PENGHANTARAN INTRASELULER EFISIEN DARI NANOPARTIKELTERAPEUTIK KE SEL KANKER PAYUDARA Oleh Ebrahim Saad Ebrahim Sadaqa NIM: 30722701 (Program Studi Doktor Farmasi) Perangkap endosomal tetapmenjadi tantangan utama dalam pengiriman intraseluler nanopartikel terapeutik, yang menyebabkan degradasi lisosom dan berkurangnya efikasi terapi. Setelah endositosis, banyak nanopartikel tetap terjebak dalam endosom dan kemudian diangkut ke jalur degradasi lisosom, mencegah pelepasan obat ke dalam sitoplasma.