Integrin Targeted Delivery of Gene Therapeutics

Integrins have become key targets for molecular imaging and for selective delivery of anti-cancer agents. Here we review recent work concerning the targeted delivery of antisense and siRNA oligonucleotides via integrins. A variety of approaches have been used to link oligonucleotides to ligands capable of binding integrins with high specificity and affinity. This includes direct chemical conjugation, incorporating oligonucleotides into lipoplexes, and use of various polymeric nanocarriers including dendrimers. The ligand-oligonucleotide conjugate or complex associates selectively with the integrin, followed by internalization into endosomes and trafficking through subcellular compartments. Escape of antisense or siRNA from the endosome to the cytosol and nucleus may come about through endogenous trafficking mechanisms, or because of membrane disrupting capabilities built into the conjugate or complex. Thus a variety of useful strategies are available for using integrins to enhance the pharmacological efficacy of therapeutic oligonucleotides

Targeted Albumin-Based Nanoparticles for Delivery of Amphipathic Drugs

We report the preparation and physical and biological characterization of human serum albumin-based micelles of approximately 30 nanometers diameter for the delivery of amphipathic drugs, represented by doxorubicin. The micelles were surface conjugated with cyclic RGD peptides to guide selective delivery to cells expressing the alpha v/beta 3 integrin. Multiple poly(ethylene glycol)s (PEGs) with molecular weight of 3400 daltons were used to form a hydrophilic outer layer, with the inner core formed by albumin conjugated with doxorubicin via disulfide bonds. Additional doxorubicin was physically adsorbed into this core to attain a high drug loading capacity, where each albumin was associated with about 50 doxorubicin molecules. The formed micelles were stable in serum but continuously released doxorubicin when incubated with free thiols at concentrations mimicking the intracellular environment. When incubated with human melanoma cells (M21+) that express the alpha v/beta 3 integrin, higher uptake and longer retention of doxorubicin was observed with the RGD-targeted micelles, than in the case of untargeted control micelles, or free doxorubicin. Consequently the RGD-targeted micelles manifested cytotoxicity at lower doses of drug than control micelles or free drug

Synthesis and evaluation of nanoglobule-cystamine-(Gd-DO3A), a biodegradable nanosized magnetic resonance contrast agent for dynamic contrast-enhanced magnetic resonance urography

Dynamic contrast-enhanced magnetic resonance imaging has been recently shown to be effective for diagnostic urography. High-resolution urographic images can be acquired with T1 contrast agents for the kidney and urinary tract with minimal noise in the abdomen. Currently, clinical contrast agents are low molecular weight agents and can rapidly extravasate from blood circulation, leading to slow contrast agent elimination through kidney and consequently providing limited contrast enhancement in urinary tract. In this study, a new biodegradable macromolecular contrast agent, nanoglobule-G4-cystamine-(Gd-DO3A), was prepared by conjugating Gd-DO3A chelates on the surface of a generation 4 nanoglobule, poly-l-lysine octa(3-aminopropyl)silsesquioxane dendrimer, via a disulfide spacer, where the carrier had a precisely defined nanosize that is far smaller than the renal filtration threshold. The in vivo contrast enhancement and dynamic imaging of the urinary tract of the agent was evaluated in nude mice using a low molecular weight agent Gd(DTPA-BMA) as a control. The agent eliminated rapidly from blood circulation and accumulated more abundantly in urinary tract than Gd(DTPA-BMA). The fast elimination kinetics is ideal for functional evaluation of the kidneys. The morphology of the kidneys and urinary tract was better visualized by the biodegradable nanoglobular contrast agent than Gd(DTPA-BMA). The agent also resulted in low liver contrast enhancement, indicating low nonspecific tissue deposition. These features render the G4 nanoglobule-cystamine-(Gd-DO3A) conjugate a promising contrast agent for magnetic resonance urography

Integrin Targeted Delivery of Gene Therapeutics

Integrins have become key targets for molecular imaging and for selective delivery of anti-cancer agents. Here we review recent work concerning the targeted delivery of antisense and siRNA oligonucleotides via integrins. A variety of approaches have been used to link oligonucleotides to ligands capable of binding integrins with high specificity and affinity. This includes direct chemical conjugation, incorporating oligonucleotides into lipoplexes, and use of various polymeric nanocarriers including dendrimers. The ligand-oligonucleotide conjugate or complex associates selectively with the integrin, followed by internalization into endosomes and trafficking through subcellular compartments. Escape of antisense or siRNA from the endosome to the cytosol and nucleus may come about through endogenous trafficking mechanisms, or because of membrane disrupting capabilities built into the conjugate or complex. Thus a variety of useful strategies are available for using integrins to enhance the pharmacological efficacy of therapeutic oligonucleotides

PhD

dissertationMagnetic resonance imaging (MRI) assists clinical diagnostics by providing high resolution images. Incorporation of contrast agents will enable MRI to detect diseases on the molecular level. The first part of the thesis describes the development and evaluation of two such contrast agents. A dendrimer-based biodegradable magnetic resonance (MR) contrast agent, PAMAM-G6-cystamine-(Gd-D03A), was developed for tumor imaging. This agent introduced higher contrast enhancement in the tumor area than the non-degradable control. Moreover, it showed faster renal excretion and lower uptake by liver tissue, suggesting favorable pharmacokinetics. The other degradable contrast agent, Nanoglobule-G4-cystamine-(Gd-D03A), was developed for dynamic contrast enhanced (DCE) MR urography. The carrier of this agent, generation 4 nanoglobule, provides a globular backbone whose size is large enough to prevent leakage in normal vasculature and small enough to allow for rapid renal elimination. The degradability, provided by disulfide bond, would further accelerate renal excretion and create a high concentration of contrast agent in the urinary tract. Images acquired by DCE-MR urography showed that this agent, although used at l/3rd the dose of Gd(DTPA-BMA), exhibited faster renal elimination kinetics, and generated significantly more contrast enhancement in the bladder than the low molecular weight Gd(DTPABMA). Meanwhile, it was rapidly removed from circulation and only allowed low liver deposition. Advances in molecular biology have conjured nucleic acids, such as plasmid DNA and siRNA, into powerful therapeutic agents. However, nucleic acid delivery exists as a paramount barrier. As a second part of the thesis, a series of multifunctional carriers (MFCs) were developed. The preliminary studies showed that one of the carriers, SKACO, could conduct luciferase expression on U87 cells up to 425 times higher than Lipofectamine-2000. This carrier also introduced green fluorescent protein expression in 71.29% of U87 cells, much higher than 6.16% by Lipofectamine-2000. Another carrier, SKAHCO, effectively delivered Luc siRNA to silence luciferase activity up to 84.6% in U87- Luc cells. It also mediated green fluorescent protein expression-knocking out in approximately 61.29% of CHO-EGFP cells, in contrast to 40.40% of Lipofectamine-2000. In summary, these carriers provide a new type of system with high delivery efficiency and are therefore worth further investigation

Integrin Targeted Delivery of Gene Therapeutics

<p>Integrins have become key targets for molecular imaging and for selective delivery of anti-cancer agents. Here we review recent work concerning the targeted delivery of antisense and siRNA oligonucleotides via integrins. A variety of approaches have been used to link oligonucleotides to ligands capable of binding integrins with high specificity and affinity. This includes direct chemical conjugation, incorporating oligonucleotides into lipoplexes, and use of various polymeric nanocarriers including dendrimers. The ligand-oligonucleotide conjugate or complex associates selectively with the integrin, followed by internalization into endosomes and trafficking through subcellular compartments. Escape of antisense or siRNA from the endosome to the cytosol and nucleus may come about through endogenous trafficking mechanisms, or because of membrane disrupting capabilities built into the conjugate or complex. Thus a variety of useful strategies are available for using integrins to enhance the pharmacological efficacy of therapeutic oligonucleotides.</p