Objectives

1. Ultrasound-Ruptured Liposomes for Enzyme-Prodrug TherapyOvercoming Dose-Limiting Side Effects of Chemotherapy AgentsMichael Benchimol (ECE), Stuart Ibsen (Bio Eng), Roy Weinstain (Pharm), Dmitri Simberg (Moores), Roger Tsien (Chem), Sadik Esener (Nano Eng) + Cleaving molecule, or Protease FluoSpheres Mothership Liposomes Macrophage Cells Benefits of Ultrasound Targeted Chemotherapy Enzyme-Prodrug Concept How it Works (The Mothership) • Motivation • Standard chemotherapy is dose-limited by its toxicity to the heart and liver • Physicians are often forced to give a sub-optimaldose, which can lead to drug resistance1 • Using approved doses still has strong side effects, weakens the immune system at a critical time, and can be fatal2 • Targeting can reduce these side effects, and kill the tumor before it becomes resistant • Challenges in • Biochemical Targeting • Tumors are biochemically different from patient to patient3 • For effective tumor targeting, the ligand expression has to be significantly greater than healthy tissue4 • Targeting tumor environment doesn’t provide necessary contrast from healthy tissue5 • Targeting relies heavily on biochemical receptor-mediated endocytosis6,7 • Ultrasound can cause a microbubble to collapse (cavitation), creating a violent shockwave and powerful jets that can shear lipid membranes • Using a nanoparticle, we can create the microbubble in situ to overcome the delivery and stability challenges of preformed microbubbles Prodrug: A drug precursor molecule which can be converted into a more toxic form by some physical or chemical means (e.g. Enzyme) β-Lactamase:A class of foreign enzymes produced by some bacteria  Needs to be protected/hidden from the immune system  Prodrugs specific forβ-Lac will not be converted by native enzymes • Safe - Non-invasive, Non-ionizing • Easy - Low cost, Little prep • Access deep tissue (For 1 MHz, 1/e @ 67 cm) • Good spatial precision • - Low tissue scattering, • - Beam focus ~ 1 mm3 • Versatile - Capable of imaging & therapy • (By tuning waveform properties) US Triggered Liposome Destruction Liposome High speed Fluorescent/US Microscope Cavitation Nanoparticle Size of Red Blood Cell Microbubble Liposome Size of nano-Mothership • Nanoscale Mothership enables: • Extravasation/better tumor penetration • Benefits from EPR Effect • Longer in vivo circulation • Greater stability, both in vivo and shelf-life Scaling Down the Particle Size Mothership Microbubble Jet of Debris Nanoparticle-based Microbubble-based After Ultrasound Before Ultrasound +Nanoliposomes are one of the only FDA-approved anti-cancer nanoparticles (Doxil) • Prodrugs can be small molecules  not dependent on endocytosis • Prodrug can be administered separately  greater control over time • Lipid-anchored enzyme easily incorporates into bilayer structures Jet from a collapsing Microbubble T. Benjamin et al., (1966) High-speed Camera Before Ultrasound After Ultrasound Need External control of payload release independent of tumor properties Fluorescently labeled β-Lactamase-lipid conjugate Payload Release (Calcein Dye) @ 1.3 MPa Microscope Objectives Liposome Stealthing with PEGylation Fluorescently Tagged Enzyme Reports its Location β-Lactamase + Lipid Membrane Incorporated β-Lactamase - Lipid Dispersed in Aqueous • To increase tumor specificity and reduce healthy tissue exposure to drugs by providing a method for external targeting independent of biochemistry • To further increase specificity by depositing exogenous enzyme to create an artificial local environment for prodrug therapy • To design a nanoscale biocompatible drug delivery vehicle (Mothership) capable of tumor penetration that contains an acoustic sensitizing nanoparticle triggered to release payload upon exposure to ultrasound Ultrasound Transducer What you don’t see here are nanoliposomes loaded with the self-quenching dye Calcein After Ultrasound Diffusion of Dye In Water, @ 2.25 MHz λ = .659 mm Ultrasound waves can be focused to achieve specificity on the mm scale Water Tank How do we get enzyme ONLY on the inner membrane leaflet? Why Size Matters Mouse Spleen (400X ) By using an engineered cleavable linker in the enzyme-lipid conjugate, the exposed enzyme can be detached and removed with a washing step A protease can also be used to digest the exposed enzyme Before Ultrasound Lipid Fragments • Cited References • Gottesman, M., “Mechanisms of Cancer Drug Resistance,” Annu. Rev. Med. 53:615–27 (2002) • Cormier, J., “Cohort Analysis of Patients With Localized, High-Risk, Extremity Soft Tissue Sarcoma Treated at Two Cancer Centers: Chemotherapy-Associated Outcomes ,” Journal of Clinical Oncology, Vol . 22, No. 22, pp. 4567-4574 (2004) • Fradet, Y., Islam, N., Boucher, L., Parent-Vaugeois, C., Tardif, M. "Polymorphic expression of a human superficial bladder tumor antigen defined by mouse monoclonal antibodies" Proc. Natl. Acad. Sci. 84, 7227-7231 (1987). • Park,J.,Hong,K.,Kirpotin, D., Colbern, G., Shalaby, R., Baselga, J., Shao, Y. , Nielsen, U., Marks, J., Moore. D., Papahadjopoulos, D., Benz, C.“ Anti-HER2 Immunoliposomes: Enhanced Efficacy Attributable to Targeted Delivery," Clinical Cancer Research, 8, 1172–1181, (2002) • Vaupel, P., Kallinowski, F., Okunieff, P., "Blood Flow, Oxygen and Nutrient Supply, and Metabolic Microenvironment of Human Tumors: A Review." Cancer Research, 49, 6449-6465 (1989) • Ulrich, A.S. “Biophysical Aspects of Using Liposomes as Delivery Vehicles.” Bioscience Reports22, 129-150 (2002). • Allen, T. "Ligand-Targeted Therapeutics in Anticancer Therapy" Nature Reviews Cancer 2 750-763 (2002) • Moghimi, S.M., Hunter, A.C. & Murray, J.C. “Long-circulating and target-specific nanoparticles: theory to practice.” Pharmacol Rev53, 283-318 (2001). • Mayer, L.D., Bally, M.B., Cullis, P.R. Strategies for Optimizing Liposomal Doxorubicin. Journal of Liposome Research 1, 463-480 (1990). Assay for Enzyme Activity Slope is proportional to enzyme activity  Lysis exposes enzyme and increases activity by 13X Intact Liposomes Red dots are accumulated micron-scale liposomes • The spleen is effective at filtering out • large particles • To achieve long circulation, the particle • should be sub-micron and malleable Enzyme can be hidden in liposome, and exposed Membrane Disruption Motherships (green) were not taken up by J774 macrophages, which could still easily phagocytose the control nanoparticles (red)