The purpose and scope of this project is subdivided in two specific aims that are detailed below:
Specific Aim 1 : Develop Multifunctional Liposomes with Targeting, Imaging and Drug Delivery Capabilities Targeting. We are constructing liposomes that target to cancer cells that over-express either folate, HER2 or CD22 receptors. Folate tethered to distearoylphosphatidylethanolamine (DSPE) via a polyethylene glycol (PEG) spacer (folatePEGDSPE) is incorporated into liposomes at 0.1-1% total lipid for targeting to folate receptors using human nasopharyngeal carcinoma (KB) cells. We have initiated imaging studies to determine biodistribution of folate-targeted fluorescent liposomes in mouse implanted with KB xenografts. For HER2 targeting we have conjugated HER2-specific Affibody (ZHER2:342-Cys, 8.3 kDa) to liposomes via its C-terminal cysteine that reacts with maleimide conjugated to pegylated phospholipid (MaL-PEG-DSPE). Fluorescent probes were incorporated into these affisomes for biophysical and/or biochemical analysis and/or triggered release assays. Affibody conjugation yields were 70% at a protein/lipid ratio of 20 g/mg with an average number of 200 affibody molecules per Affisome. Affibody conjugation did not have any significant effect on the hydrodynamic size distribution or stability of the liposomes. Affisomes are being examined for their specific interactions with HER2 molecules on human breast cancer cells (SKBR3) using fluorescence-based assays. For CD22 targeting, liposomes were constructed in a similar way using a new mutant of the CD22 scFv (HA22) with increased soluble expression (mut-HA22). The binding of mut-HA22-liposomes to CD22-expressing lymphoma cell lines (BJAB) was significantly greater than to control liposomes. Intracellular localization of mut-HA22-liposomes at 370C but not at 40C indicated that our targeted liposomes were taken up through an energy dependent process via receptor-mediated endocytosis. Mut-HA22-liposomes loaded with doxorubicin are currently being evaluated for their selective cytotoxicity to human B-lymphoma cells. Triggered Release. We have designed a novel class of light-triggerable liposomes prepared from a photo-polymerizable phospholipid DC8,9PC (1,2- bis (tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine) and DPPC (1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine). Exposure to UV (254 nm) radiation for 0-45 minutes at 25 0C resulted in photo-polymerization of DC8,9PC in these liposomes and the release of an encapsulated fluorescent dye (calcein). Kinetics and extents of calcein release correlated with mol% of DC8,9PC in the liposomes. Photopolymerization and calcein release occurred only from DPPC/DC8,9PC but not from Egg PC/DC8,9PC liposomes. These data are consistent with the notion that phase separation and packing of polymerizable lipids in the liposome bilayer are major determinants of photo-activation resulting in the formation of local defects and/or lipidic pores in the liposome membrane. This hypothesis is supported by Molecular Dynamics simulations that indicate de-mixing of DC8,9PC and DPPC in the solid phase lipid bilayer. When an appropriate tunable photo-sensitizer dye is included in the aqueous compartment of liposomes, release of contents is triggered by excitation with a laser at the wavelength of the encapsulated dye. Inhibition of release in the presence of oxygen radical scavengers indicate that the mechanism of release involves chemical changes in DC8,9PC unrelated to photo-polymerization. The laser-mediated chemical modifications in DC8,9PC are being analyzed by MS, LC, GC and NMR by the separations technology group at the advanced technology program, SAIC-Frederick. Physical characterization of DPPC: DC8,9PC liposomes include melting transition temperature (Tm), solute loading efficiency, size, and stability in serum. We are further developing these liposomes for their ability to undergo triggered release of chemotherapeutic agents (e.g. doxorubicin) and are testing their efficacy in vitro and in vivo. We are also formulating liposomes with superparamagnetic iron oxide nanoparticles (SPION) and drugs for potential applications in magnetic resonance imaging (MRI) and hyperthermia-mediated drug release.
Specific Aim 2. Development of Radiation Induced and Targeted Chemotherapy (RITCH). The concept envisions a non-toxic pro-drug that when administered intravenously will distribute throughout the body. When the pro-drug is subjected to localized electromagnetic radiation it will undergo a chemical transformation into a cytotoxic compound at the site of the tumor. We have used as a prototype the hydrophobic membrane probe Iodonaphthyl-azide (INA), which upon light irradiation undergoes a covalent reaction with transmembrane portions of membrane proteins. Photo-activation of INA affects the signaling capabilities of numerous cellular receptors and results in cell death. Since the INA treatment eliminates multidrug transporter function in addition to other membrane proteins, this approach is advantageous for treatment of multidrug resistant tumors. The unique mechanism of action INA targeting membrane proteins thus provides a novel and potent chemotherapeutic approach. Recently we observed that alternate radiation modalities (e.g. sono-cavitation and X-ray radiation) can trigger the reactivity of RITCH compounds. We plan to examine efficacy of our RITCH compounds in vitro and in vivo using various modes of triggering that include light, sono-cavitation and X-ray radiation The animal studies involve pharmacokinetics, bio-distribution and toxicity using the small animal imaging facility at NCI-Frederick. We are designing new RITCH compounds that will be more amenable to various triggering modes. In addition we are pursuing basic studies on the chemistry of the new compounds as well on cell biological events that lead to apoptosis and cell death.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010652-06
Application #
8157381
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
6
Fiscal Year
2010
Total Cost
$786,733
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Afonin, Kirill A; Viard, Mathias; Martins, Angelica N et al. (2013) Activation of different split functionalities on re-association of RNA-DNA hybrids. Nat Nanotechnol 8:296-304
Kim, Taejin; Afonin, Kirill A; Viard, Mathias et al. (2013) In Silico, In Vitro, and In Vivo Studies Indicate the Potential Use of Bolaamphiphiles for Therapeutic siRNAs Delivery. Mol Ther Nucleic Acids 2:e80
Yavlovich, Amichai; Viard, Mathias; Gupta, Kshitij et al. (2013) Low-visibility light-intensity laser-triggered release of entrapped calcein from 1,2-bis (tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine liposomes is mediated through a type I photoactivation pathway. Int J Nanomedicine 8:2575-87
Sinthuvanich, Chomdao; Veiga, Ana Salomé; Gupta, Kshitij et al. (2012) Anticancer ?-hairpin peptides: membrane-induced folding triggers activity. J Am Chem Soc 134:6210-7
Lee, Jae-Ho; Oh, Hyuntaek; Baxa, Ulrich et al. (2012) Biopolymer-connected liposome networks as injectable biomaterials capable of sustained local drug delivery. Biomacromolecules 13:3388-94
Puri, Anu; Jang, Hyunbum; Yavlovich, Amichai et al. (2011) Material properties of matrix lipids determine the conformation and intermolecular reactivity of diacetylenic phosphatidylcholine in the lipid bilayer. Langmuir 27:15120-8
Smith, Brandon; Lyakhov, Ilya; Loomis, Kristin et al. (2011) Hyperthermia-triggered intracellular delivery of anticancer agent to HER2(+) cells by HER2-specific affibody (ZHER2-GS-Cys)-conjugated thermosensitive liposomes (HER2(+) affisomes). J Control Release 153:187-94
Puri, Anu; Blumenthal, Robert (2011) Polymeric lipid assemblies as novel theranostic tools. Acc Chem Res 44:1071-9
Yavlovich, Amichai; Singh, Alok; Blumenthal, Robert et al. (2011) A novel class of photo-triggerable liposomes containing DPPC:DC(8,9)PC as vehicles for delivery of doxorubcin to cells. Biochim Biophys Acta 1808:117-26
Colantonio, Simona; Simpson, John T; Fisher, Robert J et al. (2011) Quantitative analysis of phospholipids using nanostructured laser desorption ionization targets. Lipids 46:469-77

Showing the most recent 10 out of 20 publications