Lee Josephson

Here, we report on NBD-Wm, a fluorescent wortmannin (Wm) probe that maintains the bioactivity of Wm as an inhibitor of PI3 kinase and as an antiproliferative agent. The attachment of the NBD fluorochrome permits NBD-Wm in cells to be monitored by NBD fluorescence-based methods such as FACS or fluorescence microscopy or with an anti-NBD antibody. The fluorescence of NBD-Wm treated cells reached a peak at 1.5 h and then decreased because of the extrusion of a fluorescent compound into the culture media. Cells accumulated NBD-Wm to levels about 30-fold higher than those in the media. NBD-Wm modified five major proteins, with the modification of the catalytic subunit of PI3 kinase being a minor band. The bioactivity of NBD-Wm, coupled with a variety of techniques available for determining its disposition, suggest that NBD-Wm can be a useful tool in understanding the mechanism of action of viridins.

We synthesized three peptides, aD‐polyarginyl peptide (r8(FITC)), a Tat peptide (Tat(FITC)), and a control peptide (Cp(FITC)) and attached each to amino‐CLIO, a nanoparticle 30 nm in diameter. We then examined the effective permeability,P_eff, of all six materials through CaCo‐2 monolayers. The transport of peptide–nanoparticles was characterized by a lag phase (0–8 h) and a steady‐state phase (9–27 h). The steady‐stateP_effvalues for peptides were in the order r8(FITC)>Tat(FITC)=Cp(FITC). When r8(FITC) and Tat(FITC) peptides were attached to the nanoparticle, they conferred their propensity to traverse cell monolayers onto the nanoparticle, whereas Cp(FITC) did not. Thus, when the r8(FITC) peptide was attached to the amino‐CLIO nanoparticle, the resulting peptide–nanoparticle had aP_effsimilar to that of this poly‐d‐arginyl peptide alone. TheP_effof r8(FITC)–CLIO (M_W∼1000 kDa) was similar to that of mannitol (M_W=182 Da), a poorly transported reference substance, with a far lower molecular weight. These results are the first to indicate that the modification of nanoparticles by attachment of membrane‐translocating sequence‐based peptides can alter nanoparticle transport through monolayers. This suggests that the surface modification of nanoparticles might be a general strategy for enhancing the permeability of drugs and that high‐permeability nanoparticle‐based therapeutics can be useful in selected pharmaceutical applications.

The many uses of chemically modified annexin Vs necessitate an understanding of the optimal degree of modification and modification sites of the protein. When reacted with the N‐hydroxysuccinimide ester of Cy5.5, annexin V with one modification per mole of protein retained its affinity for phosphatidylserine of apoptotic cells, whereas modification with two dyes per mole of protein caused a complete loss of activity. A tryptic digest LC/MS method was used to identify the modification sites as either of two closely spaced lysine residues, in position 286 or 290. The crystal structure indicated the location of these lysines was distal to the phosphatidylserine binding sites on annexin V. These results can be used to develop active or inactive fluorescent control annexin V proteins and to suggest strategies for attaining higher levels of modification with retention of bioactivity.

A number of different types of agents have been employed to aid in the visualization of lymph nodes, particularly the sentinel lymph node, and to decrease the tissue destruction associated with the diagnosis of nodal metastases. The current study was performed to see if a novel macromolecular near-infrared fluorescent (NIRF) probe could be used to visualize lymph nodes after intravenous administration (pan-node visualization) or subcutaneous administration (sentinel node visualization), and serve as method for guiding dissection with interventional radiologic and surgical procedures. Cy5.5-PGC, the near-infrared dye Cy5.5 coupled to a protected graft copolymer (PGC), was injected (i.v. or s.c.) into nude mice. Twenty-four hours later white light and NIRF images were obtained on (i) the live animal, (ii) a partially dissected animal, and (iii) tissue specimens. With Cy5.5-PGC administered intravenously, axillary nodes were visualized from outside a living mouse. With partial dissection, iliac and aortic nodes were visible as concentrated foci of high-intensity NIRF signals. With subcutaneous injection in the front extremity, axillary and brachial nodes draining the injection site were easily visualized. NIRF imaging provides a nonradioactive method of visualizing lymph nodes through layers of tissue that can be employed with intravenous or subcutaneous injection.

A number of different types of agents have been employed to aid in the visualization of lymph nodes, particularly the sentinel lymph node, and to decrease the tissue destruction associated with the diagnosis of nodal metastases. The current study was performed to see if a novel macromolecular near-infrared fluorescent (NIRF) probe could be used to visualize lymph nodes after intravenous administration (pan-node visualization) or subcutaneous administration (sentinel node visualization), and serve as method for guiding dissection with interventional radiologic and surgical procedures. Cy5.5-PGC, the near-infrared dye Cy5.5 coupled to a protected graft copolymer (PGC), was injected (iv or sc) into nude mice. Twenty-four hours later white light and NIRF images were obtained on (i) the live animal, (ii) a partially dissected animal, and (iii) tissue specimens. With Cy5.5-PGC administered intravenously, axillary nodes were visualized from outside a living mouse. With partial dissection, iliac and aortic nodes were visible as concentrated foci of high-intensity NIRF signals. With subcutaneous injection in the front extremity, axillary and brachial nodes draining the injection site were easily visualized. NIRF imaging provides a nonradioactive method of visualizing lymph nodes through layers of tissue that can be employed with intravenous or subcutaneous injection.