Paclitaxel-Loaded Expansile Nanoparticles Enhance Chemotheraputic Drug-delivery in Mesothelioma 3D Multicellular Spheroids
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1 Paclitaxel-Loaded Expansile Nanoparticles Enhance Chemotheraputic Drug-delivery in Mesothelioma 3D Multicellular Spheroids Hongyi Lei 1, Rong Liu 1, Aaron Colby 2, Mark W. Grinstaff 2, Yolonda L. Colson 1 1 Brigham and Women s Hospital, Boston, MA 2 Boston University, Boston, MA April 29, 2014, Toronto
2 None Disclosure
3 Challenges in current evaluation of local chemotherapeutic drug delivery systems Efficacy of chemotherapy against tumors are often surprisingly different between in vitro and in vivo studies. Several studies have shown that experiments with monolayer cell cultures do not predict in vivo results with animal models or clinical outcomes Unfortunately, in vivo studies are costly particularly for purposes of drug screening and mechanism testing. There has been significant interest in the development of more relevant in vitro models of tumor growth and structure, including several different tumor spheroid models.
4 Pax-C/E superior to Pax-eNP in human malignant mesothelioma cytotoxicity testing in vitro Paclitaxel-loaded expansile nanoparticles (Pax-eNP) are less effective (i.e. higher IC50) than an equivalent concentration of free paclitaxel (Pax-C/E) in a monolayerbased cytotoxicity assays in vitro. Pax-C/E IC50= 12.4 Pax-eNP IC50= 36.9 Colson YL, et al. Biomaterials, 2011
5 Pax-eNP superior to Pax-C/E in the local treatment of peritoneal mesothelioma in vivo Local Pax-eNP treatment of established mesothelioma in an in vivo animal model significantly improves survival compared to equivalent Pax-C/E dosing Overall survival (%) 100 Pax-eNP (n=12) Pax-C/E (n=12) Unloaded enp (n=6) # Days post xenograft Median Survival (days) : P < vs Pax-C/E or Unloaded enp #: P < vs. Unloaded enp Doubling of survival merely as a function of enp drug delivery
6 Can in vitro 3D mesothelioma spheroids model the Pax-eNP enhanced cytotoxicity noted in vivo? Hypothesis: Superior efficacy of Pax-eNP in vivo is secondary to early, persistent tumor penetration and prolonged intratumoral release of chemotherapeutic drug? 3D Spheroid Model Studies of drug and nanoparticle tumor penetration require multicellular models 5000 MSTO-211H human mesothelioma cells Multicellular spheroid models shown to be more resistant to chemotherapy Spheroids form within 24 hrs without collagen or hydrogel
7 Rho enp uptake increases in mesothelioma tumor spheroids over time Rho-eNP 2 µg/ml, 12h 2 µg/ml, 24h 2 µg/ml, 48h 2 µg/ml, 72h Macropinocytosis (wortmannin) is the dominant endocytotic pathway for enp uptake Rho-eNP Inhibitor Inhibition of Rho-eNP (100%) Wortmannin NaN 3 +2-D-G PI3K inhibitor Methyl Sodium channel (energy dependent) Amiloride Chlorpromazine Selective ion channel inhibitor Cholesterol-dependent pathway clathrin-mediated caveolae-mediated Genistein Rho-eNP Control Macropinocytosis P < 0.05 vs. Rho-eNP Control
8 Enhanced paclitaxel delivery via enp induces Caspase 3 in tumor spheroids OGPax-eNP Pax-rho-eNP OG-Pax intensity (10 5 ) OGPax uptake in spheroid at 24 hrs : P < 0.05 vs OGPax-C/E OGPax-eNP OGPax-C/E Caspase-3 intensity Pax-eNP induces Caspase-3 : P < 0.05 vs 12 and 24hrs # : P < 0.05 vs 48 hrs 12h 24h 48h 72h Time (hour) # 12h 24h 48h 72h Spheroids treated with Pax-rho-eNP (red) demonstrate caspase-3 (green) upregulation
9 Pax delivery via enp yields greater tumor cytotoxicity against mesothelioma spheroids 4/24 4/48 4/72 Pax-eNP or Pax-C/E Pax-eNP 4hrs Wash away drug Pax-C/E Spheroids in drug-free culture media 24, 48, or 72 hrs PI intensity (10 6 ) # Pax-C/E Pax-eNP : P < 0.05 vs. Pax-C/E 4/72 # : P < 0.05 vs. Pax-eNP 4/48 Confocal imaging (PI) Assess Cell Death 0.0 4/24 4/48 4/72 Drug exposure/post-exposure Time(h)
10 Conclusions Compared to standard monolayer cell culture, this in vitro 3D tumor spheroid culture model better reflects the increased tumor cytotoxicity demonstrated by Pax-eNP in vivo. 3D spheroid models can be valuable tools for investigating cytotoxic mechanisms and nanoparticle-tumor interactions particularly given high costs or limitations of in vivo animal studies. Potential use of spheroids as a clinically relevant platform for screening novel pharmaceuticals and unique drug-delivery systems during the early pre-clinical development phase.
11 Future Studies Investigate: Architectural-based mechanism by which tumors demonstrate drug resistance in vivo - i.e. why do cells develop resistance when organized as spheroids enp-tumor cell interactions & biologic response to injury in a unique tissue spheroid construct containing a macrophage-tumor cell-collagen complex that mimics tumor architecture in vivo Acknowledgements: Laboratories of Dr. Yolonda Colson at BWH and Dr. Mark Grinstaff at BU Mesothelioma Applied Research Foundation and IMP program at BWH.
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