Modular Functionalities At The Biosynthetic Interface 521 Targeting

The ability to physically target diseased cells to receive therapeutics while avoiding residual uptake in other tissues has long been a goal in cancer therapy.9-12 The homing of stem cells to a tissue niche, or the susceptibility of one cell over another to viral infection, demonstrates that biomole-cular recognition can be used to direct species to specific extracellular and intracellular sites. The microenvironment of the tumor including cell-surface markers, extracellular matrix, soluble factors, and proteases, as well as the tumor's unique architecture and transport properties, may be exploited for targeting.13-17

Both passive and active targeting have been utilized for nanoparticle delivery. Passive targeting relies upon the unique pharmacokinetics of nanoparticles including minimal renal clearance and enhanced permeability and retention (EPR) through the porous angiogenic vessels in the tumor.18,19 Surface attachment of polymers such as poly(ethylene glycol) (PEG) and poly(ethylene oxide) (PEO) enables nanoparticles to avoid uptake by mononuclear phagocytes in the liver, spleen, and lymph nodes, thereby improving accumulation in the tumor.20-22 Active targeting relies on ligand-directed binding of nanoparticles to receptors expressed in the tumor. Binding of ligands to the vasculature can occur immediately, as it is directly accessible to nanoparticles circulating in the blood. Over longer time periods, particles extravasate into the tissues where receptors expressed on cancer cells and in the interstitium may be used for localization.23-25

Many candidate tumor markers have been described, some of which bind known ligands such as arginine-glycine-aspartic acid (RGD)-binding Vb3 and Vb5 integrins expressed on the surface of angiogenic blood vessels, and folic acid-binding receptors on the surface of cancer cells. These and others have been attached to the surface of various nanoparticle cores to deliver them to

17 26_28

tumors. , Monoclonal antibodies have also been used extensively for targeting. These can be isolated with high affinity for tumor markers and are useful for targeting receptors of unknown or low affinity ligands.29,30 Novel screens for discovering tumor homing ligands have been developed using phage and bacterial display as well as libraries of aptamers, peptides, polymers, and small molecules.31,32 These techniques may be used to isolate targeting ligands, even when their target receptor is unknown. For example, the 34 amino acid, cationic peptide F3, which has been used to deliver quantum dots to tumor endothelium, was uncovered initially by a blind-page display screen in a breast cancer xenograft model and later found to bind cell surface nucleolin expressed on tumor endothelium and cancer cells.6,33,34

Although extracellular targeting to the tumor is sufficient for many modes of imaging and drug delivery, intracellular delivery of nanoparticles into the cytosol is essential for some applications. For example, nanoparticles carrying membrane-impermeable cargo that perform their function in the cytosol, such as siRNA, antisense DNA, peptides, and other drugs, are minimally effective if delivered extracellularly or sequestered in the endosome.35 Protein and peptide motifs capable of translocating nanoparticles into the cytoplasm have been borrowed from mechanisms of viral transfection. Two important classes of translocating domains include polycationic sequences and membrane fusion domains. Attaching the short polycationic sequence of HIV's TAT protein, amino acid residues 48-57, to a nanoparticle facilitates its adsorption on a cell surface and subsequent internalization into the cell.36,37 This peptide has been used to internalize dextran coated iron-oxide nanoparticles into T-cells in vitro, which were subsequently used to monitor T-cell trafficking in tumors with MRI.38 Use of this peptide for intracellular delivery in vivo is limited by the adverse effect that polycationic sequences have on nanoparticle circulation time and RES uptake.39 The amphiphilic domain derived from the N-terminus of the influenza protein hemagluttinin (HA2) is a membrane fusion peptide that destabilizes the endosome at low pH and facilitates viral escape into the cytosol.40 Variations of this peptide with improved infectivity have also been synthesized.41 Influenza-derived peptides have been used to enhance the delivery of liposomes as well as 100 nm poly-l-lysine particles. Although the peptide modification of these particles improves endosomal escape over unmodified particles, the transfection efficiency still remains well below that of intact


viruses. ,

Another level of targeting can occur after translocation of nanoparticles into the cytosol to direct nanoparticles to specific sub-cellular structures. Using peptide localization sequences, fluorescent quantum dots have been targeted to the nucleus and the mitochondria (Figure 5.2).2 Several other localization sequences exist and could be used to traffic nanoparticles to the endoplasmic reticulum, golgi apparatus, or peroxisomes. Although work in this area has been focused on organelle labeling, the potential for delivering therapeutic nanoparticles to sub-cellular structures is possible. Such nanoparticles could sense sub-cellular aspects of disease or specifically intervene for more potent treatment or eradication of cancer cells (i.e., free-radical-mediated mitochondrial damage to induce apoptosis).

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