Group members: Martin Latterich, Philippe Valadon, Adrian Chrastina, Malgorzata Czarny, Yanzheng Liu, Phil Oh
This facility develops, optimizes, and tests a range of carefully controlled, multifunctional nanoparticles with different physical properties and links them to targeting antibodies. Our lab currently works with gold-, silver-, and silica-based nanoparticles, dendrimers and dendrimer fragments called dendrons, nanostreptabodies, and liposomes. We are also part of active collaborations with a local nanotechnology company, nanoComposix, to develop novel nanoparticles and with Dr. Shuming Nie of Emory University to test next-generation Quantum Dots in vivo. Each of these nanoparticles is likely to have different behaviors in vivo. Thus, nanoparticle stability, interactions with blood components, the reticulo-endothelial system, and endothelium, and degradation and excretion must be compared to determine the most appropriate nanoparticle for clinical applications. This facility works closely with the imaging facility to create nanoparticles that can actively target specific organs or disease states in vivo.
The Nanoparticle Development and Production facility currently provides PRISM investigators with access to a Submicron particle analyzer, a Buchi Rotary evaporator, a LabConco Freezone Freeze Dryer, a dark field/bright field/fluorescent Olympus BX41 optical microscope coupled to a 10 megapixel color CCD camera, 2 Agilent 8453 diode spectrophotometers, a Hitachi F-4500 fluorescent spectrophotometer, a PE AAnalyst 200 atomic absorption spectrophotometer, a Brookfield viscometer, a Malvern Zetasizer Nano ZS dynamic light scattering instrument, a Horiba Static Light Scattering Instrument, a Tantec Contact Angle Instrument, a Varian Tap Density Meter, a Mettler liquid density meter, and a Mathis TCi thermal conductivity meter, a Biorobotics Microgrid II microarrayer with 96 slide capacity, a JEOL 1010 100kV transmission electron microscope equipped with a 2K x 2K XR41B AMT digital camera, Hitachi S5000 SEM, a FTS Dura-Stop microprocessor controlled freeze drying system, a Vacuum Atmospheres inert atmosphere glovebox and aerosol characterization chambers. This facility also has full access to the complete PRISM lab space, which includes cold rooms, radioactive labeling preparation and analysis rooms, tissue culture suites, communal instrument rooms, scintillation counters, refrigerators, chemistry hoods ultracentrifuges and key rotors, tissue culture incubators ultra-low freezers, a MilliQ water purification system, and general-use computers.
Dendrimers:
Dendrimers are highly branched, well-defined, monodisperse polymers with a relatively uniform size and molecular weight that are synthesized through a series of reactions (generations) that add branched reactive groups. Each generation has a distinct size. Commercially available dendrimers are readily available from 1-10 nm, and the final size can be increased by adding functional moieties to surface of the dendrimer. In our laboratory, cargo can be attached to dendrimers through encapsulation in the hollow dendritic core, absorption on to the surface through electrostatic interactions, association with the PEGylated surface, or through chemical conjugation to functional groups at the surfaces of the dendrimer. The generation 5 dendrimers commonly used in our lab have 128 terminal nitrogen groups that can be used to covalently conjugate antibodies, imaging agents, or drugs to the dendrimers. Both pH sensitive linkages (through hydrazone, phosphoramidate, ortoester, and vinylester bonds) and reducible linkages (through disulfide bonds) can be used to covalently attach drugs. Because of their highly branched structure and abundance of functional groups, dendrimers can carry multiple payloads to create novel, multifunctional NPs.
Unfunctionalized polyamidoamine (PAMAM) dendrimers are normally rapidly scavenged from the blood by the RES and can be toxic due to the cationic surface. We and others find that surface derivatization of the dendrimer surface or the addition of inert, hydrophilic residues such as PEG, to the surface of dendrimers helps to mask these charges, reduces haemotoxicity effects and RES uptake, increases the ability of drug molecules to be adsorbed onto the surface of the dendrimer, and may help protect drugs from interaction with serum proteins.
Colloidal gold NPs have been used for several decades because their high electron density and relatively simple conjugation to proteins make them ideal markers for use in biological imaging. Gold-based NPs are generally made by the reduction of HAuCl4 with an appropriate reductant. Great control can be obtained over the size, shape, and morphology of the gold-based NPs by adjusting the conditions of the NP growth. Proteins can be bound to the surface of metallic NPs through electrostatic interactions with the positively charged NP surface, through hydrophobic interactions, and/or through direct covalent interaction of free sulfhydryl groups of the protein to the gold surface. Additionally, direct covalent coupling of proteins to the gold through an appropriate chelating linker can be employed. This facility routinely attached avidin covalently to the surface of the gold, allowing for “tinker toy” attachment of a variety of biotinylated targeted antibodies, imaging agents, and effector molecules to create multifunctional NPs.
Lipid-based NPs:
Lipid-based NPs are spherical vesicles (~30 nm - >1 mm) comprised of a phospholipid-based bilayer membrane. This biphasic system contains an internal aqueous core and a lipid outer membrane that can encapsulate and deliver a broad spectrum of hydrophobic, amphipathic, and hydrophilic compounds. Lipid-based NPs have been extensively studied and successfully used for delivery of drugs and imaging agents and even for DNA transfer as a non-viral gene carrier. Lipid-based NPs are biocompatible carriers (due to their biodegradability, low toxicity and non-immunogenicity). They also can reduce potential toxicity associated with free drugs.
