Full-length TNC and TNCIII1–5 cross-competition ELISA.

<p>Testing of binding of full length TNC to a selection of growth factors and cross-competition binding assay of TNC with TNCIII1–5. (A) Binding of TNC (10 nM) to PDGF-BB, BDNF, VEGF-A165, FGF-2, and BMP-2. (B) An increasing dose of full length TNC (0 to 50 nM) resulted in higher binding levels of TNC to PDGF-BB (black). No specific binding to BSA (grey) was observed. (C–G) Cross-competition assay between full length TNC and TNCIII1–5 (10 nM) to bind selected growth factors: PDGF-BB (C), BDNF (D), VEGF-A165 (E), FGF-2 (F), and BMP-2 (G). The TNC concentration varied from 0.1 nM to 50 nM (n = 3, mean±SD), with 100% representing binding of 10 nM TNCIII1–5 without the presence of TNC.</p

Identification of growth factors with an affinity towards TNCIII1–5.

<p>Binding of TNCIII1–5 to growth factors of different families, measured using indirect ELISA. Binding was measured in the presence of 4 µM heparin (grey) and in its absence (black). Various growth factor families were tested, PDGF/VEGF family (A), FGF family (B), TGF-β family (C), IGF family and IGF-BPs (D), and others (E) (n = 3, mean±SD).</p

The effect of heparin concentration on the ability of TNCIII1–5 to bind selected growth factors.

<p>Using ELISA, the binding of TNCIII1–5 (10 nM) to various growth factors (A) PDGF-BB, (B) VEGF-A165, (C) FGF-2, and (D) BDNF was measured in the presence of increasing concentrations of heparin, 0.01 nM to 10,000 nM, with 100% representing binding of 10 nM TNCIII1–5 without the presence of heparin. The binding of the growth factors to TNCIII1–5 reached a maximum at an intermediate heparin concentration for all growth factors tested (n = 3, mean±SD).</p

Determination of the specific growth factor-binding domain.

<p>The binding affinity of different (fusion) domains (GST-TNCIII3, GST-TNCIII4, GST-TNCIII5, GST-TNCIII3-4, GST-TNCIII4-5, GST-TNCIII3-5) to PDBF-BB was determined and compared to controls, GST only and BSA, using an indirect ELISA method. The TNC domain responsible for growth factor binding was mainly identified as domain TNCIII5, with TNCIII4 contributing to a lesser extent. (n = 3, mean±SD).</p

Equilibrium binding constants (K_D), and association (k_on) and dissociation (k_off) rates were determined using surface plasmon resonance (SPR) and fitting the data to Langmuir binding kinetics for binding of immobilized TNCIII1–5 to TGF-β1 and NT-3.

*<p>Not applicable (NA) because the data was not interpretable using standard Langmuir binding kinetics for FGF-2 and PDGF-BB.</p

Production and characterization of TNCIII1-5.

<p>(A) Top: schematic of structural domains of full length human tenascin C (TNC), with major integrin binding and heparin binding domains. Bottom: schematic of the designed TNCIII1-5 fragment. (B) SDS-PAGE gel of purified TNCIII1–5 with the molecular weight of 53 kD, and the different GST-TNCIII (fusion) domains (GST-TNCIII3, GST-TNCIII4, GST-TNCIII5, GST-TNCIII3-4, GST-TNCIII4-5, GST-TNCIII3-5). (C) Amino acid sequence of TNCIII1–5. (D) Graphical depiction of isoelectric points of the TNCIII domains.</p

Reduction-Sensitive Tioguanine Prodrug Micelles

Colloidal drug and prodrug conjugates have unique targeting characteristics for tumor vasculature from the blood and for the lymphatics draining a tissue injection site. Tioguanine and tioguanine-generating prodrugs have been investigated as anticancer and immunosuppressive agents, including use in cancer immunotherapy. Recently we developed block copolymers of poly­(ethylene glycol)-<i>bl</i>-poly­(propylene sulfide) that self-assemble in aqueous solutions to form micellar structures. Since the polymers carry a free terminal thiol group resulting from the ring-opening polymerization of the propylene sulfide monomer, we sought to prepare prodrug block copolymers with tioguanine linked by a reduction-sensitive disulfide bond. The synthesis involved a disulfide exchange between the oxidized form of tioguanine and the polymer. Spectroscopic data is presented to support the proposed reaction. The polymers self-assembled when dispersed in water to form tioguanine prodrug micelles with a size range between 18 and 40 nm that released tioguanine in response to cysteine and serum as shown spectroscopically. In comparison with a poly­(ethylene glycol) prodrug polymer, we show that the rate of tioguanine release can be controlled by changing the poly­(propylene sulfide) block length and that the tioguanine remains bioactive with cultured cells

Preparation of Well-Defined Ibuprofen Prodrug Micelles by RAFT Polymerization

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used to treat acute pain, fever, and inflammation and are being explored in a new indication in cancer. Side effects associated with long-term use of NSAIDs such as gastrointestinal damage and elevated risk of stroke, however, can limit their use and exploration in new indications. Here we report a facile method to prepare well-defined amphiphilic diblock copolymer NSAID prodrugs by direct reversible addition–fragmentation transfer (RAFT) polymerization of the acrylamide derivative of ibuprofen (IBU), a widely used NSAID. The synthesis and self-assembling behavior of amphiphilic diblock copolymers (PEG-PIBU) having a hydrophilic poly­(ethylene glycol) block and a hydrophobic IBU-bearing prodrug block were investigated. Release profiles of IBU from the micelles by hydrolysis were evaluated. Furthermore, the antiproliferative action of the IBU-containing micelles in human cervical carcinoma (HeLa) and murine melanoma (B16–F10) cells was assessed

Human clonal urothelial cells arising from single ureteral cell.

<p>(A) Schematics showing the passage from the single selected cell to the in vivo implantated clonal cell pellet. (B,C and D) Human urothelial holoclone, meroclone and paraclone cultures arising from one single ureteral cell. (E and F) Growth curve of a human urothelial holoclone (G) <i>In vivo</i> urothelial differentiation of human ureteral urothelial holoclone pellets implanted into the subcapsular space of the Swiss nu/nu mice, expressing cytokeratin 7, uroplakin-2 and uroplakin 3 (scale bars, 10 µm). Note the “micro-bladder” like structure.</p

Porcine clonal urothelial cells arising from a single urethral cell.

<p>(A, B and C) Porcine urethelial holoclone, meroclone and paraclone cultures arising from a single urethral cell. (D and E) Growth curves of a porcine urothelial holoclone. (F, G) <i>In vivo</i> urothelial differentiation of porcine urethral urothelial holoclonal cell pellets implanted into the subcapsular space of the Swiss nu/nu mice, expressing cytokeratin 7, uroplakin-2 and uroplakin 3 (scale bars, 10 µm). Note the “micro-bladder” like structure.</p