c39d609577b1422756bc946bffd03bcb pmc_10668968.pdf 0965713f21a1d6e48f7b337cf26658c447c6f2e1 pmc_10668968.pdf 4886e527942eff0631867ea3844568c0fd0c8b04e5e8178410a37bfe9806ddec pmc_10668968.pdf Title: Lenvatinib-Loaded Poly(lactic-co-glycolic acid) Nanoparticles with Epidermal Growth Factor Receptor Antibody Conjugation as a Preclinical Approach to Therapeutically Improve Thyroid Cancer with Aggressive Behavior Subject: Background: Lenvatinib, a tyrosine kinase inhibitor (TKI) approved for the treatment of progressive and radioactive iodine (RAI)-refractory differentiated thyroid cancer (DTC), is associated with significant adverse effects that can be partially mitigated through the development of novel drug formulations. The utilization of nanoparticles presents a viable option, as it allows for targeted drug delivery, reducing certain side effects and enhancing the overall quality of life for patients. This study aimed to produce and assess, both in vitro and in vivo, the cytotoxicity, biodistribution, and therapeutic efficacy of lenvatinib-loaded PLGA nanoparticles (NPs), both with and without decoration using antibody conjugation (cetuximab), as a novel therapeutic approach for managing aggressive thyroid tumors. Methods: Poly(lactic-co-glycolic acid) nanoparticles (NPs), decorated with or without anti-EGFR, were employed as a lenvatinib delivery system. These NPs were characterized for size distribution, surface morphology, surface charge, and drug encapsulation efficiency. Cytotoxicity was evaluated through MTT assays using two cellular models, one representing normal thyroid cells (Nthy-ori 3-1) and the other representing anaplastic thyroid cells (CAL-62). Additionally, an in vivo xenograft mouse model was established to investigate biodistribution and therapeutic efficacy following intragastric administration. Results: The NPs demonstrated success in terms of particle size, polydispersity index (PDI), zeta potential, morphology, encapsulation efficiency, and cetuximab distribution across the surface. In vitro analysis revealed cytotoxicity in both cellular models with both formulations, but only the decorated NPs achieved an ID50 value in CAL-62 cells. Biodistribution analysis following intragastric administration in xenografted thyroid mice demonstrated good stability in terms of intestinal barrier function and tumor accumulation. Both formulations were generally well tolerated without inducing pathological effects in the examined organs. Importantly, both formulations increased tumor necrosis; however, decorated NPs exhibited enhanced parameters related to apoptotic/karyolytic forms, mitotic index, and vascularization compared with NPs without decoration. Conclusions: These proof-of-concept findings suggest a promising strategy for administering TKIs in a more targeted and effective manner. Keywords: thyroid; nanoparticles; EGFR; lenvatinib Author: Giovanna Revilla, Nuseibah Al Qtaish, Pablo Caruana, Myriam Sainz-Ramos, Tania Lopez-Mendez, Francisco Rodriguez, Verónica Paez-Espinosa, Changda Li, Núria Fucui Vallverdú, Maria Edwards, Antonio Moral, José Ignacio Pérez, Juan Carlos Escolà-Gil, José Luis Pedraz, Idoia Gallego, Rosa Corcoy, María Virtudes Céspedes, Gustavo Puras and Eugènia Mato Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Tue Nov 14 12:46:15 2023 CET ModDate: Wed Nov 15 02:23:25 2023 CET Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 19 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 4784078 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- QJLLGS+VnURWPalladioL Type 1 Custom yes yes no 10 0 ONYKLE+URWPalladioL-Bold Type 1 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Materials Destination: subsection.2.1 Item: Title: Preparation of Lenvatinib-Loaded PLGA Nanoparticles (Lenva-NPs) Destination: subsection.2.2 Item: Title: Characterization of Lenva-NPs: Particle Size, Polydispersity Index (PDI), Zeta Potential Measurements, and Morphology Studies (TEM) Destination: subsection.2.3 Item: Title: Transmission Electron Microscope (TEM) Studies Destination: subsection.2.4 Item: Title: Assessing the Incorporation of Lenvatinib into Nanoparticles with HPLC Destination: subsection.2.5 Item: Title: In Vitro Lenvatinib Release Studies Destination: subsection.2.6 Item: Title: Preparation of Immunonanoparticles (Lenva-NPs-Cetuximab) Destination: subsection.2.7 Item: Title: Characterization of Lenvatinib-NP-Cetuximab Immunonanoparticles Destination: subsection.2.8 Item: Title: Quantification of Cetuximab Conjugated onto the Surface of Nanoparticles Destination: subsection.2.9 Item: Title: Preparation of DilC 18 Fluorescently Labeled Nanoparticles Destination: subsection.2.10 Item: Title: Cell Lines Destination: subsection.2.11 Item: Title: In Vitro Cytotoxicity Studies of Lenva-NPs and Lenva-NP-Cetux Destination: subsection.2.12 Item: Title: Biodistribution of NP and NP-Cetux Nanoparticles in a Mouse Xenograft Model Destination: subsection.2.13 Item: Title: Mouse Xenograft Model: In Vivo Intragastric Administration of Nanoparticles with Lenvatinib and Nanoparticles with Lenvatinib and Decorated with Cetuximab Destination: subsection.2.14 Item: Title: Histopathological and Histomorphometric Assessment and Immunohistochemistry Analysis of Tumor Necrosis and Proliferation Rates Destination: subsection.2.15 Item: Title: Western Blotting Destination: subsection.2.16 Item: Title: Statistical Analysis Destination: subsection.2.17 Item: Title: Results Destination: section.3 Children: Item: Title: Physicochemical Characterization and In Vitro Release Profile of Lenva-NPs and Lenva-NPs-Cetuximab Destination: subsection.3.1 Item: Title: Physicochemical Characterization of DilC 18-NPs and DilC 18-NPs-Cetuximab Destination: subsection.3.2 Item: Title: In Vitro Cytotoxicity Studies of Lenva-NPs and Lenva-NPs-Cetuximab Destination: subsection.3.3 Item: Title: Biodistribution of Lenva-NPs and Lenva-NPs-Cetuximab Destination: subsection.3.4 Item: Title: In Vivo Evaluation of the Therapeutic Effect of Lenva-NPs and Lenva-NPs-Cetux Destination: subsection.3.5 Item: Title: Discussion Destination: section.4 Item: Title: References Destination: section.A. Info: Title: Lenvatinib-Loaded Poly(lactic-co-glycolic acid) Nanoparticles with Epidermal Growth Factor Receptor Antibody Conjugation as a Preclinical Approach to Therapeutically Improve Thyroid Cancer with Aggressive Behavior Author: Giovanna Revilla, Nuseibah Al Qtaish, Pablo Caruana, Myriam Sainz-Ramos, Tania Lopez-Mendez, Francisco Rodriguez, Verónica Paez-Espinosa, Changda Li, Núria Fucui Vallverdú, Maria Edwards, Antonio Moral, José Ignacio Pérez, Juan Carlos Escolà-Gil, José Luis Pedraz, Idoia Gallego, Rosa Corcoy, María Virtudes Céspedes, Gustavo Puras and Eugènia Mato Subject: Background: Lenvatinib, a tyrosine kinase inhibitor (TKI) approved for the treatment of progressive and radioactive iodine (RAI)-refractory differentiated thyroid cancer (DTC), is associated with significant adverse effects that can be partially mitigated through the development of novel drug formulations. The utilization of nanoparticles presents a viable option, as it allows for targeted drug delivery, reducing certain side effects and enhancing the overall quality of life for patients. This study aimed to produce and assess, both in vitro and in vivo, the cytotoxicity, biodistribution, and therapeutic efficacy of lenvatinib-loaded PLGA nanoparticles (NPs), both with and without decoration using antibody conjugation (cetuximab), as a novel therapeutic approach for managing aggressive thyroid tumors. Methods: Poly(lactic-co-glycolic acid) nanoparticles (NPs), decorated with or without anti-EGFR, were employed as a lenvatinib delivery system. These NPs were characterized for size distribution, surface morphology, surface charge, and drug encapsulation efficiency. Cytotoxicity was evaluated through MTT assays using two cellular models, one representing normal thyroid cells (Nthy-ori 3-1) and the other representing anaplastic thyroid cells (CAL-62). Additionally, an in vivo xenograft mouse model was established to investigate biodistribution and therapeutic efficacy following intragastric administration. Results: The NPs demonstrated success in terms of particle size, polydispersity index (PDI), zeta potential, morphology, encapsulation efficiency, and cetuximab distribution across the surface. In vitro analysis revealed cytotoxicity in both cellular models with both formulations, but only the decorated NPs achieved an ID50 value in CAL-62 cells. Biodistribution analysis following intragastric administration in xenografted thyroid mice demonstrated good stability in terms of intestinal barrier function and tumor accumulation. Both formulations were generally well tolerated without inducing pathological effects in the examined organs. Importantly, both formulations increased tumor necrosis; however, decorated NPs exhibited enhanced parameters related to apoptotic/karyolytic forms, mitotic index, and vascularization compared with NPs without decoration. Conclusions: These proof-of-concept findings suggest a promising strategy for administering TKIs in a more targeted and effective manner. 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