Ponatinib (Pon) is a multi-tyrosine kinase inhibitor that demonstrated high efficiency for treating cancer. However, severe side effects caused by Pon off-targeting effects prevent its extensive use. Using our understanding into the mechanisms by which Pon is transported by bovine serum albumin in the blood, we have successfully encapsulated Pon into a biomimetic nanoparticle (NP). This lipid NP (i.e., “leukosomes”) incorporates membrane proteins purified from activated leukocytes that enable immune evasion, and enhanced targeting of inflamed endothelium NPs have been characterized for their size, charge, and encapsulation efficiency. Membrane proteins enriched on the NP surface enabled modulation of Pon release. These NP formulations showed promising dose–response results on two different murine osteosarcoma cell lines, F420 and RF379. Our results indicate that our fabrication method is reproducible, nonuser-dependent, efficient in loading Pon, and applicable toward repurposing numerous therapeutic agents previously shelved due to toxicity profiles.

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Osteosarcoma (OS) is the most common pediatric bone tumor with a worldwide incidence of 3.4 cases per million people annually. Unfortunately, current treatments are still not sufficient to eradicate OS, due to its ability to resist the upfront standard chemotherapy. Therefore, it is essential to identify and effectively deliver novel therapeutic regimens. Multi-tyrosine kinase inhibitors have been explored as new therapeutics for different sarcomas. Among many, Ponatinib (Pon) demonstrated very potent anti-tumor activity, however, it received a black box warning from the FDA due to significant cardiovascular side effects. Recently, our lab developed novel biomimetic nanoparticles (NPs) called Leukosomes (Leuko) capable of encapsulating and effectively releasing Pon. These NPs are synthesized from activated leukocytes, maintaining leukocytes' tropism towards inflamed endothelium. Leveraging on this technology, we aim to validate the therapeutic potential of Leuko in 3D OS tumor model (sarcospheres) and their ability to target primary murine OS model while concomitantly reducing the detrimental side effects. Given its biological relevance and ability to better recapitulate the tumor structure, a 3D tumor model was chosen to reproduce key properties, such as diffusion limitations and the cellular network of solid tumors that have significant impacts on cancer drug efficacy.In the sarcospheres model, we observed efficient penetration and internalization of NPs in both, murine (RF379, 577) and human PDX derived (PDX94, pPDX202) OS cell lines, where Leuko exhibited a 20% increase in targeting vs the control Liposome (Lipo). This difference was not detected in the control 2D model. Moreover, our data demonstrated a 2-fold increase in the Leuko cytotoxic effect in 3D compared to 2D systems. Murine OS cell viability was 20% lower in sarcospheres after treatment with the IC50 for Pon. In addition, NPs induced complete inhibition of murine sarcosphere formation in the extreme limiting dilution assay (ELDA). These findings were also confirmed using the PDX derived OS cell lines. Subsequently, an in vivo intratibial syngeneic orthotopic mouse model was utilized to determine the targeting and biodistribution of the NPs. Leuko showed an increased targeting and penetration in the tumor 1 and 3h post NPs injection as anticipated by the 3D in vitro studies. By exploiting the inflammatory conditions within the tumor, which increases the Leuko accumulation, these preliminary results advocate the translational potential of this innovative formulation as a new targeted drug delivery approach for OS. This promising platform could improve the clinical therapeutic approaches and lead to improved outcomes and reduced side effects for OS patients. Citation Format: Federica Giordano, Stefania Lenna, Riccardo Rampado, April Ewing, Gherardo Baudo, Matteo Massaro, Assaf Zinger, Enrica De Rosa, Jason T Yustein, Francesca Taraballi. Ponatinib loaded leukocyte-based nanoparticles for osteosarcoma treatment in sarcosphere tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 311.

The pro‐inflammatory microenvironment that contributes to atherosclerotic plaque progression is sustained by M1 macrophages. Metabolic reprogramming toward heightened glycolysis accompanies M1 macrophage polarization, with approaches aimed at lessening glycolytic metabolism in macrophages standing to impact disease progression. The objective is to decrease the inflammatory response in atherosclerotic lesions by inducing favorable metabolic phenotypes in macrophages using an innovative mitochondrial transplantation strategy. The hypothesis is that delivery of mitochondria, functionalized with a dextran and triphenylphosphonium (Dextran‐TPP) polymer conjugate for enhanced cellular transplantation, to atherosclerotic plaques properly regulates M1 macrophage bioenergetics, attenuating inflammatory processes and preventing plaque progression. Dextran‐TPP mitochondria transplantation to M1 macrophages has profound effects on cell bioenergetics, resulting in increased oxygen consumption rate and reduced glycolytic flux that coincides with a decreased inflammatory response. Upon intravenous delivery to ApoE−/− mice fed a high fat diet, Dextran‐TPP mitochondria accumulate in aortic plaques and co‐localize with macrophages. Importantly, Dextran‐TPP mitochondria treatment reduces the plaque burden in ApoE−/− mice, improving cholesterol levels, and ameliorating hepatic steatosis and inflammation. Findings highlight Dextran‐TPP mitochondria as a novel biological particle for the treatment of atherosclerosis, underlining the potential for macrophage metabolic regulation as a therapy in other diseases.

The introduction of targeted anticancer drugs has revolutionized treatment. Multi-tyrosine kinase inhibitors (TKIs) are potential therapeutics targeting specific signaling pathways that contribute to cancer progression, including sarcomas. Osteosarcoma is the most common pediatric tumor with a worldwide incidence of 3.4 cases/million annually but without effective treatment. Of the TKIs, Ponatinib demonstrated potent anti-tumor activity; however, it received an FDA black box warning for potential side effects. New treatment and delivery systems must be identified to use Ponatinib clinically. Our laboratory developed novel biomimetic nanoparticles (NPs) synthesized from activated leukocytes called Leukosomes. Leukosomes maintain leukocyte tropism towards inflamed endothelium and can encapsulate and effectively release Ponatinib. We aimed to validate Leukosome technology’s therapeutic potential to specifically target and inhibit primary murine osteosarcoma growth and reduce detrimental side effects. In a 3D sarcosphere model, we observed effective penetration and internalization of NPs in both murine (RF379, 577) and human patient derived xenograft (PDX94, PDX202) osteosarcoma cell lines. Leukosome exhibited 20% increased targeting versus control liposomes. Cell viability decreased 20% after treatment with Ponatinib IC50. Leukosome-Ponatinib IC50 also induced complete inhibition of murine sarcosphere formation and ~60-80% reduction of cell viability. We developed an osteosarcoma orthotopic mouse model by intratibial injection of murine F420 osteosarcoma cells. After 3 weeks, tumor was detected and the mice were injected with Ponatinib NPs. Leukosome showed increased tumor targeting and penetration 1 and 3 hours post-NP injection. Intravenous tail injection of Ponatinib was lethal due to drug-related toxic effects, while intraperitoneal injection of Ponatinib was well tolerated. Conversely, intravenous injection of Ponatinib-loaded NPs did not show toxic effects. In vivo efficacy studies demonstrated that 3 weeks of Ponatinib treatment (2 treatments/week) inhibited tumor growth and increased survival. Similar results were also observed after Leukosome Ponatinib treatment, despite 10 times less Ponatinib in NPs than in free drug. Overall, these results show that leukocyte-derived membrane proteins enhance the accumulation of Ponatinib at the tumor site and surrounding inflamed tissue. While limited Ponatinib encapsulation in NPs remains an open challenge, this formulation underlies the possibility of reducing drug dosage and side effects. Thus, suggest Leukosome’s translational potential as a new targeted drug delivery approach with better outcomes and fewer complications for osteosarcoma patients. Citation Format: Federica Giordano, Stefania Lenna, Riccardo Rampado, Gherardo Baudo, Matteo Massaro, Ashley Rivera, Enrica De Rosa, Jason T. Yustein, Francesca Taraballi. Ponatinib loaded leukocyte-based nanoparticles: A new platform for treating osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 307.

Small-molecule tyrosine kinase inhibitors (TKIs) represent a potentially powerful approach to the treatment of osteosarcoma (OS). However, dose-limiting toxicity, therapeutic efficacy, and targeting specificity are significant barriers to the use of TKIs in the clinic. Notably among TKIs, ponatinib demonstrated potent anti-tumor activity; however, it received an FDA black box warning for potential side effects. We propose ponatinib-loaded biomimetic nanoparticles (NPs) to repurpose ponatinib as an efficient therapeutic option for OS. In this study, we demonstrate enhanced targeting ability and maintain potent ponatinib nano-therapeutic activity, while also reducing toxicity. In in vitro two- and three-dimensional models, we demonstrate that ponatinib-loaded biomimetic NPs maintain the efficacy of the free drug, while in vivo we show that they can improve tumor targeting, slow tumor growth, and reduce evidence of systemic toxicities. Though there is limited Pon encapsulation within NPs, this platform may improve current therapeutic approaches and reduce dosage-related side effects to achieve better clinical outcomes in OS patients. Graphical Abstract