Michael D. Kennedy

Folate-targeted enzyme prodrug cancer therapy utilizing penicillin-V amidase and a doxorubicin prodrug.

In antibody-targeted enzyme prodrug therapy, a monoclonal antibody (mAb) covalently linked to an enzyme is commonly exploited to concentrate the enzyme on the tumor cell surface prior to administration of a relatively nontoxic prodrug. The tumor-localized enzyme then converts the prodrug into a cytotoxic agent, which in turn diffuses into the tumor causing localized cell death. In this paper, we have substituted folic acid for the mAb as a mean of delivering an attached enzyme, penicillin-V amidase (PVA), to folate receptor (FR)-positive tumor cells. The enzyme PVA is capable of converting a doxorubicin-N-p-hydroxyphenoxyacetamide prodrug (DPO) into its potent parent drug, doxorubicin. For PVA targeting, each PVA molecule was covalently labeled with three molecules of folic acid via the formation of amide bonds. In vitro binding assays showed that folate-PVA-125I conjugates bind specifically to KB cells (FR-positive tumor cells) but not to A549 cells (FR-negative tumor cells). Moreover, in a series of in vitro cytotoxicity tests, folate-PVA conjugates were found to kill folate receptor positive but not receptor negative cells, and when bound to FR-positive cells, folate-PVA conjugates rendered the DPO prodrug as toxic as free doxorubicin (IC50, approximately 0.6 microM). Finally, preliminary in vivo plasma clearance studies in normal mice revealed that i.v. administered folate-PVA-125I and PVA-125I are both cleared from the blood within a 24 h time period, removing concern that nonspecifically trapped folate-PVA might activate prodrug in nontargeted tissues. In view of the fact that only a small number of folate-PVA molecules are required to mediate killing of target cells in vitro, these data argue that folate-targeted enzyme prodrug therapy should be considered for tumor eradication in vivo.

Optical imaging of metastatic tumors using a folate-targeted fluorescent probe

We describe the use of a tumor targeting ligand, the vitamin folic acid, to deliver an attached fluorescent probe to both primary and metastatic tumors overexpressing the folate receptor. Upon laser excitation, derived images of normal tissues generally show little or no fluorescence, whereas images of folate receptor-expressing tumors display bright fluorescence that can be easily distinguished from adjacent normal tissue. Furthermore, metastatic tumor loci of submillimeter size can also be visualized without the aid of image processing or enhancement. The sharp distinction between tumor and normal tissues provided by this technique could find application in the localization and resection of tumor tissue during surgery or in the enhanced endoscopic detection and staging of cancers.

Evaluation of folate conjugate uptake and transport by the choroid plexus of mice

Purpose. Because the choroid plexus (CP) is enriched in cell surface folate receptors, an investigation was initiated to evaluate whether folate receptor-mediated transcytosis might be exploited to deliver folate conjugates into the brain.Methods. Balb/c mice were injected with radioactive and fluorescent conjugates of folate to measure and image their uptake by the CP.Results. Retention of a radioactive folate conjugate, folate-diethylenetriaminepentaacetic acid (DTPA)-111In, into the brain of balb/c mice was observed, although repeated injections or prolonged release via an osmotic pump of the compound did not result in increased brain uptake. Uptake of an 125I-labeled anti-folate receptor antibody into the brain was very low, and no competition was observed with unlabeled antibody. Imaging of brain thin-sections and whole brain tissue from a mouse injected with folate-fluorescein revealed strong fluorescence in the CP, but virtually no where else in the brain.Conclusions. Both fluorescence and radioimaging results demonstrate specific uptake of small molecular weight folate conjugates into CP cells of the murine brain, but no significant transport of the molecules across the CSF. Furthermore, no uptake of larger folate-linked proteins by choroid plexus cells is observed, suggesting folate conjugate size may strongly influence access to CP folate receptors.

Riboflavin-mediated delivery of a macromolecule into cultured human cells

Cell surface receptors for the vitamins folic acid and biotin have been previously reported to mediate the endocytosis of vitamin-conjugated macromolecules into cultured cells. To evaluate whether a similar uptake pathway for riboflavin-conjugated macromolecules might exist, riboflavin was covalently linked to bovine serum albumin (BSA) via the vitamins ribityl side chain, and uptake of the protein by cultured human cells was examined. Whereas unconjugated BSA was not internalized by KB, A549, SK-LU-1 or SK-OV cells, riboflavin-conjugated BSA was readily internalized (>106 molecules/cell). Analysis of the uptake pathway revealed that the riboflavin-BSA conjugate likely docks on cells at a carrier/transport protein that is distinct from the uptake pathway for free riboflavin and then enters via normal membrane cycling. Evidence for this contention is: (i) the internalized conjugate accumulates in endosomal compartments, (ii) uptake into cells is halted at temperatures near 0 degreesC where membrane trafficking is abrogated, (iii) cell association is inhibited by unlabeled riboflavin-BSA, but not by free riboflavin, and (iv) cellular uptake of [3H]riboflavin is only partially inhibited by riboflavin-BSA. Regardless of the pathway of internalization, these data demonstrate that riboflavin conjugation can facilitate protein entry into human cells in culture.

Increased uptake of folate conjugates by activated macrophages in experimental hyperlipemia

In the pathogenesis of atherosclerosis, macrophages become activated and play a crucial role in plaque formation. Activated synovial macrophages have recently been shown to express receptors for folic acid. We have determined whether activated macrophages also over-express folate receptor (FR) in atherosclerosis. Most normal cells express little or no FR, and, if FR is present on activated macrophages, folate-linked compounds and drugs could be selectively targeted to those cells that do express FR. To evaluate the FR on macrophages of atherosclerotic animals, golden Syrian hamsters were maintained on a hyperlipidemic diet until extensive vascular lesions had developed. Uptake of folic acid conjugated to fluorescent tags was then examined in tissue fragments from lesion-prone areas, and peritoneal activated macrophages were harvested from the same animals. Spectrofluorimetric and fluorescence microscopic analyses showed a significantly greater uptake of folate-conjugates by peritoneal macrophages of hyperlipidemic hamsters compared with those of hamsters fed a normal or folate-deficient diet. Systemically administered folate-fluorescent conjugates were found to accumulate as bright spots in protrusions of atherosclerotic plaques populated by macrophages, whereas a low level of fluorescence was detected uniformly dispersed across the lesion. The uptake of the folate conjugate by U937 macrophage cells grown in a high-lipid culture medium was significantly higher than in controls. Our data thus indicate that hyperlipidemic conditions induce an increased uptake of folate attributable to the over-expression of FRs on activated macrophages. This increase in FR expression can be exploited to deliver folate-linked compounds selectively to atherosclerotic lesions.

Statistical approach for detection and localization of a fluorescing mouse tumor in Intralipid

A statistical approach is presented for detecting and localizing a fluorescing tumor obscured underneath millimeters of turbid medium. The method is validated in an experimental study using a mouse tumor and folate-iridocyanine dye.

Erythrocyte adhesion is modified by alterations in cellular tonicity and volume

We tested the hypothesis that dehydration‐induced alterations in red blood cell (RBC) membrane organisation or composition contribute to sickle cell adhesion in sickle cell disease (SCD). To examine the role of RBC hydration in adhesion to the subendothelial matrix protein thrombospondin‐1 (TSP), normal and sickle RBCs were incubated in buffers of varying tonicity and tested for adhesion to immobilised TSP under flow conditions. Sickle RBCs exhibited a decrease in TSP binding with increasing cell hydration (P < 0·005), suggesting that cellular dehydration may contribute to TSP adhesion. Consistent with this hypothesis, normal RBCs showed an increase in TSP adhesion with increasing dehydration (P < 0·01). Furthermore, increased TSP adhesion of normal RBCs could also be induced by isotonic dehydration using nystatin‐sucrose buffers. Finally, TSP adhesion of both sickle RBCs and dehydrated normal RBCs was inhibited by the anionic polysaccharides, chondroitin sulphate A and high molecular weight dextran sulphate, but not by competitors of CD47‐, band 3‐, or RBC phosphatidylserine‐mediated adhesion. More importantly, we found increased adhesion of nystatin‐sucrose dehydrated normal mouse RBCs to kidney capillaries following re‐infusion in vivo. In summary, these findings demonstrate that changes in hydration can significantly impact adhesion, causing normal erythrocytes to display adhesive properties similar to those of sickle cells and vice versa.

Ion-assisted deposition of oxide materials at room temperature by use of different ion sources.

Thin films of SiO(2), TiO(2), Ta(2)O(5), ZrO(2), and the mixed oxide H4 (Merck) have been deposited onto nonheated glass substrates by electron-beam evaporation in commercial coating plants. All depositions have been carried out with ion assistance provided by three different ion or plasma sources (end-hall, plasma, and cold-cathode sources). The optical film properties such as index of refraction, extinction coefficient, light scattering, and absorption have been examined by spectrophotometry, laser calorimetry, and total integrated light-scatter measurements. Surface morphology has been investigated by atomic force microscopy studies. Furthermore, films have undergone sand erosion tests for the determination of relative wear resistance. The film properties are compared for the three different ion sources.

Ion-assisted deposition processes: industrial network IntIon

The presented work is embedded in the research network “Integrative Ion Processes for Modern Optics”, called IntIon, consisting of 12 partners from the German optics industry and two research institutes. The main target of the IntIon network is the development of new process concepts on the basis of ion assisted deposition (IAD) for the industrial production of optical thin film components. Besides an improvement in efficiency, a major aim is concentrated on the optical characteristics for selected application fields with high economical potential. In this network, different ion and plasma sources are compared with regard to their qualification for ion assisted deposition processes. This work includes the characterization of the ion energy and ion current using Faraday-cup measurements. The selection of investigated coating materials includes a broad variety of standard and non-standard oxides. First results of the network will be presented for adapted deposition materials and different operation characteristics of ion sources.

IAD of oxide coatings at low temperature: a comparison of processes based on different ion sources

A comparative study of different ion and plasma assisted physical vapor deposition processes at low temperature is reported. To work out a clear comparison of the different processes, the object of the study are single layers of different metal oxides like Ta2O5, TiO2, SiO2 and mixed oxides like H4 (Merck) deposited on glass and silicon substrates. Three different types of ion- (or plasma-respectively) sources are used: the cold cathode ion source from Denton (CC 104), the end hall ion source Mark II from CSC and the advanced plasma source from Leybold. Each of these processes is run under conditions concerning process parameters like bias, ion current, ion energy, beam characteristics and gas flow, which were understood to be optimized also to maintain long-term stability as realistic production conditions. The resulting metal oxide single layers are characterized by their optical properties, dispersion curves for NUV and VIS as well as absorption and scatter at discrete wavelengths. Also discussed are mechanical properties like hardness and adherence. A test method is presented which clearly shows the superior behavior of the IAD coatings.