Nanoparticles have opened new exciting avenues for both diagnostic and therapeutic applications in human disease, and targeted nanoparticles are increasingly used as specific drug delivery vehicles. The precise quantification of nanoparticle internalization is of importance to measure the impact of physical and chemical properties on the uptake of nanoparticles into target cells or into cells responsible for rapid clearance. Internalization of nanoparticles has been measured by various techniques, but comparability of data between different labs is impeded by lack of a generally accepted standardized assay. Furthermore, the distinction between associated and internalized particles has been a challenge for many years, although this distinction is critical for most research questions. Previously used methods to verify intracellular location are typically not quantitative and do not lend themselves to high throughput analysis. Here we developed a mathematical model which integrates the data from high throughput flow cytometry measurements with data from quantitative confocal microscopy. The generic method described here will be a useful tool in biomedical nanotechnology studies. The method was then applied to measure the impact of surface coatings of vesosomes on their internalization by cells of the reticuloendothelial system (RES). RES cells are responsible for rapid clearance of nanoparticles, and the resulting fast blood clearance is one of the major challenges in biomedical applications of nanoparticles. Coating of vesosomes with long chain polyethylene glycol showed a trend for lower internalization by RES cells.

Means to prevent thrombus extension and local recurrence remain suboptimal, in part because of the limited effectiveness of existing thrombolytics. In theory, plasminogen activators could be used for this purpose if they could be anchored to the vascular lumen by targeting stably expressed, noninternalized determinants such as platelet-endothelial-cell adhesion molecule 1 (PECAM-1). We designed a recombinant molecule fusing lowmolecular-weight single-chain prourokinase plasminogen activator (lmw-scuPA) with a single-chain variable fragment (scFv) of a PECAM-1 antibody to generate the prodrug scFv/lmw-scuPA. Cleavage by plasmin generated fibrinolytically active 2-chain lmw-uPA. This fusion protein (1) bound specifically to PECAM-1-expressing cells; (2) was rapidly cleared from blood after intravenous injection; (3) accumulated in the lungs of wild-type C57BL6/J, but not PECAM-1 null mice; and (4) lysed pulmonary emboli formed subsequently more effectively than lmw-scuPA, thereby providing support for the concept of thromboprophylaxis using recombinant scFv-fibrinolytic fusion proteins that target endothelium. ( IntroductionPlasminogen activators (PAs; eg, uPA, urokinase plasminogen activator) help to restore perfusion after thrombotic vascular occlusion, the leading cause of human morbidity and mortality. [1][2][3] However, the clinical utility of PAs is limited by (1) inadequate delivery because of rapid elimination and inactivation en route and ineffective penetration into formed clots; (2) side effects, including extravasation leading to collateral damage in the central nervous system and other tissues; (3) lysis of "physiologic" (hemostatic) clots leading to hemorrhage; and, (4) reperfusion injury following a delay in restoring perfusion, where morbidity correlates with the duration of ischemia. [4][5][6] Clinical settings characterized by a high propensity for thrombosis have been identified, and means to diagnose early clot formation have been developed. 1,2 Although the indications for prophylaxis are known, PAs are not used prophylactically because of their unfavorable pharmacokinetics and side effects. Gene therapy approaches, effective in cell-culture and animal experiments, 7,8 are not practical when the need to enhance fibrinolysis is acute and of short duration. 9 Conceivably, prophylactic delivery of a PA derivative that rapidly restricts and sustains its activity in the vascular lumen can help to lyse nascent clots expeditiously, inhibit propagation of mural thrombi, and reduce the duration of ischemia.For example, PAs can be used for thromboprophylaxis by coupling to carrier red blood cells (RBCs), prolonging circulation and limiting extravasation. 10 This approach may have utility in settings in which RBC transfusion is part of current management. Drug targeting to suitable endothelial-cell-surface determinants 11-13 may provide an alternative approach and, in theory, localize PA activity in the affected intravascular compartment.For example, drugs coupled with antibodies to plateletendothelial ...

Targeting the tumor vasculature and selectively modifying endothelial functions is an attractive anti-tumor strategy. We prepared polyethyleneglycol modified immunoliposomes (IL) directed against vascular cell adhesion molecule 1 (VCAM-1), a surface receptor over-expressed on tumor vessels, and investigated the liposomal targetability in vitro and in vivo. In vitro, anti-VCAM-1 liposomes displayed specific binding to activated endothelial cells under static conditions, as well as under simulated blood flow conditions. The in vivo targeting of IL was analysed in mice bearing human Colo 677 tumor xenografts 30 min and 24 h post i.v. injection. Whereas biodistribution studies using [3H]-labelled liposomes displayed only marginal higher tumor accumulation of VCAM-1 targeted versus unspecific ILs, fluorescence microscopy evaluation revealed that their localisations within tumors differed strongly. VCAM-1 targeted ILs accumulated in tumor vessels with increasing intensities from 30 min to 24 h, while control ILs accumulated in the tumor tissue by passive diffusion. ILs that accumulated in non-affected organs, mainly liver and spleen, primarily co-localised with macrophages. This is the first morphological evidence for selective in vivo targeting of tumor vessels using ILs. VCAM-directed ILs are candidate drug delivery systems for therapeutic anti-cancer approaches designed to alter endothelial function.