Paul O. Zamora

Cell attachment and biocompatibility of polytetrafluoroethylene (PTFE) treated with glow-discharge plasma of mixed ammonia and oxygen

The plasma generated from a gas mixture of NH3 plus O2 (NH3 + O2) has been used to impart unique chemical and biological characteristics to polytetrafluoroethylene (PTFE). PTFE treated with NH3 + O2 plasma was physiochemically distinct from surfaces treated with plasma of either NH3 or O2 alone, as determined by electron spectroscopy for chemical analysis (ESCA). The contact angle analysis revealed that the PTFE surfaces became less hydrophobic after plasma treatments. ESCA results indicate the presence of oxygen-containing groups and nitrogen-containing groups at the plasma-treated surfaces. PTFE treated with NH3 + O2 plasma resisted the attachment of platelets and leukocytes in a manner similar to untreated PTFE; however, the attachment of bovine aorta endothelial cells was substantially increased. Once attached, these cells grew to confluency. The increased endothelial cell attachment was higher than that observed following plasma treatment with each gas used separately, which could be attributed to the considerable amount of CF(OR)2-CF2 formed on the NH3 + O2 plasma-treated PTFE surface. At 14 days after subcutaneous implantation in rats, the PTFE wafers treated with NH3 + O2 plasma demonstrated less encapsulation and lower levels of inflammatory cells compared to controls. Collectively, the results suggest that NH3 + O2 plasma treatment imparts a unique character to PTFE and could be useful in certain in vivo applications.

Experimental radiotherapy of receptor‐positive human prostate adenocarcinoma with 188Re‐RC‐160, a directly‐radiolabeled somatostatin analogue

The therapeutic potential of the somatostatin analogue RC‐160 radiolabeled with 188Re was evaluated in nude mice bearing xenografts of human prostate adenocarcinoma. 188Re‐RC‐160 was selectively retained in both DU‐145 and PC‐3 tumors following direct intra‐tumor injection at all time points examined (2, 6 and 24 hr post‐injection). Unbound 188Re‐RC‐160 was rapidly excreted via the hepatobiliary system and, with the exception of the gastrointestinal tract, very little normal organ uptake was found at any time point examined. Negative control compounds, 188Re‐perrhenate and 188Re‐mercaptoacetyl‐triglycine (188Re‐MAG3), were essentially washed out of the tumor by 6 hr post‐injection and were rapidly excreted through the kidneys. 131I‐RC‐160, used as a reference compound, had a biodistribution in tumor‐bearing animals similar to that of 188Re‐RC‐160. In PC‐3 xenografts, 188Re‐RC‐160 gave a dose‐dependent therapeutic response (stasis or regression) even in animals with relatively large tumor masses (greater than 600 mm3), whereas the macro‐aggregated form of 188Re‐RC‐160 did not. Long‐term studies with 188Re‐RC‐160 demonstrated a protracted reduction of tumor volume and a positive effect on animal survival. Neither RC‐160 by itself nor a 188Re‐labeled peptide, unrelated to somatostatin (PA‐22‐2, a laminin peptide), demonstrated the reduction in tumor mass observed with 188Re‐RC‐160. 188Re‐RC‐160 shows potential as a new clinical agent for treatment of somatostatin‐receptor‐positive cancers.

Comparison of fluorine-18 fluorodeoxyglucose positron emission tomography and technetium-99m methoxyisobutylisonitrile scintimammography in the detection of breast tumours

The aim of this study was to compare, in breast cancer patients, the diagnostic accuracy of positron emission tomography (PET) using fluorine-18 fluorodeoxyglucose (FDG) and scintimammography (SMM) using technetium-99m methoxyisobutylisonitrile (MIBI). A total of 20 patients (40 breasts with 22 lesions) were evaluated serially with MIBI and, on the following day, with FDG. For SMM, planar and single-photon emission tomography imaging in the prone position was performed starting at 10 min following the injection of MIBI (740 MBq). For PET, scans were acquired 45–60 min after the injection of FDG (370 MBq) and attentuation correction was performed following transmission scans. Results from SMM and PET were subsequently compared with the histopathology results. True-positive results were obtained in 12/13 primary breast cancers (mean diameter=29 mm, range 8–53 mm) with both FDG and MIBI. False-negative results were obtained in two local recurrences (diameter <9 mm) with both FDG and MIBI. In benign disease, FDG and MIBI did not localize three fibrocystic lesions, two fibroadenomas and one inflammatory lesion (true-negative), but both localized one fibroadenoma (false-positive). Collectively, the results demonstrate a sensitivity of 92%, and a specificity of 86%, for primary breast cancer regardless of whether FDG or MIBI was used. In contrast to MIBI scintigraphy, FDG PET scored the axillae correctly as either positive (metastatic disease) or negative (no axillary disease) in all 12 patients. The tumour/non-tumour ratio for MIBI was 1.97 (range 1.43–3.1). The mean standard uptake value (SUV) for FDG uptake was 2.57 (range 0.3–6.2). The diagnostic accuracy of SMM was equivalent to that of FDG PET for the detection of primary breast cancer. For the detection of in situ lymph node metastases of the axilla, FDG seems to be more sensitive than99mTc-MIBI.

A synthetic, bioactive PDGF mimetic with binding to both α-PDGF and β-PDGF receptors

A multi-domain peptide, PAB2-1c, was designed and synthesized as a bioactive mimic of PDGF. PBA2-1c bound to both α- and β-PDGF receptors as determined by surface plasmon resonance (SPR). The equilibrium dissociation constant (Kd) of binding to α-PDGF receptors by PAB2-1c (1.7 × 10− 8 M) compared favorably rhPDGF-AA (1.34 × 10− 8 M). Binding to β-PDGF receptor by PAB2-1c (2.2 × 10− 8 M) was less favorable than, that of recombinant human PDGFBB (1.59 × 10− 9 M). Interestingly, PBA2-1c bound to these two receptors with similar affinity suggesting that, PBA2-1c was not PDGF receptor selective. In a murine myoblast cell line C2C12, PBA2-1c increased the tyrosine phosphorylation on PDGF receptors and the phosphorylation of AKT and ERK1/2 in a concentration-related manner. PBA2-1c also stimulated an increase in cell proliferation, cell migration, and collagen gel contraction. In these cell-based assays, PAB2-1c was effective at 1 μg/ml or lesser. The results support the hypothesis that, PBA2-1c is a mimetic of PDGF, although it has a more promiscuous receptor interaction.

Controlled Drug Release through a Plasma Polymerized Tetramethylcyclo-tetrasiloxane Coating Barrier

A plasma polymerized tetramethylcyclo-tetrasiloxane (TMCTS) coating was deposited onto a metallic biomaterial, 316 stainless steel, to control the release rate of drugs, including daunomycin, rapamycin and NPC-15199 (N-(9-fluorenylmethoxy-carbonyl)-leucine), from the substrate surface. The plasma-state polymerized TMCTS thin film was deposited in a vacuum plasma reactor operated at a radio-frequency of 13.56 MHz, and was highly adhesive to the stainless steel, providing a smooth and hard coating layer for drugs coated on the substrate. To investigate the influence of plasma coating thickness on the drug diffusion profile, coatings were deposited at various time lengths from 20 s to 6 min, depending on the type of drug. Atomic force spectroscopy (AFM) was utilized to characterize coating thickness. Drug elution was measured using a spectrophotometer or high-performance liquid chromatography (HPLC) system. The experimental results indicate that plasma polymerized TMCTS can be used as an over-coating to control drug elution at the desired release rate. The drug-release rate was also found to be dependent on the molecular weight of the drug with plasma coating barrier on top of it. The in vitro cytotoxicity test result suggested that the TMCTS plasma coatings did not produce a cytotoxic response to mammalian cells. The non-cytotoxicity of TMCTS coating plus its high thrombo-resistance and biocompatibility are very beneficial to drug-eluting devices that contact blood.

Localization of Small‐cell Lung Cancer Xenografts with Iodine‐125‐, Indium‐111‐, and Rhenium‐188‐Somatostatin Analogs

We examined the potential of radiolabeled somatostatin analogs, 125I‐Tyr‐3‐octreotide (125I‐octreotide), 111In‐DTPA(diethylenetriaminepentaacetatic acid)‐d‐Phe‐1‐octreotide (111In‐octreotide), and 188Re‐octreotide for targeting small‐cell lung cancer (SCLC) in a mouse model. Tyr‐3‐octreotide was labeled with 125I by the chloramine T method, and 111In‐octreotide was obtained as a kit, while 188Re was eluted from a 188W/188Re generator, and octreotide was directly labeled with 188Re by reducing disulfide bonds. The 125I‐, 111In‐, and 188Re‐octreotides were injected i.v. into athymic mice bearing NCI‐H69 tumors, and the biodistributions were determined at 15 min, and 2, 4, 8, and 24 h. Tumor uptakes were 0.5±0.2, 0.3±0.1, 0.3±0.1 %ID/g, and tumor‐to‐blood ratios were 1.8, 11.9, 1.2 at 8 h for 125I‐, 111In‐, and 188Re‐octreotides, respectively. Accumulations of 111In‐octreotide in normal tissues were lower than those of 125I‐ and 188Re‐octreotides. 188Re‐octreotide can be used to localize SCLC lesions as efficiently as radioiodinated octreotide. However, 111In‐octreotide was the most suitable agent to obtain high tumor‐to‐normal tissue contrast for localizing SCLC.

B2A as a positive BMP receptor modulator

B2A (B2A2-K-NS) is a synthetic multi-domain peptide that in vitro augments bone morphogenetic protein (BMP)-2-induced cell responsiveness and osteodifferentiation. Augmentation of endogenous BMP-2 is thought to ultimately improve bone repair, and has led to clinical evaluation of B2A in orthopedic applications. In this study, we show that B2A binds to BMP receptor (BMPR)-IB, BMPR-II, and BMPR-IA. B2A reduces the EC50 of rh-BMP-2, thus shifting the response curve to the left. B2A enhances the osteogenic activity of BMP-2, but not growth and differentiation factor-5, BMP-7, or BMP-9, indicating its action is highly BMP-2 selective. Additionally, B2A did not augment Wnt-3a- and retinoic acid-induced differentiation. All three functional domains (receptor-binding domain, hydrophobic-linker domain, heparin-binding domain) of B2A are required for optimal bioactivity. Collectively, the results suggest that B2A, via its unique sequence, acts in a manner consistent with a positive receptor modulator to selectively enhance BMP-2 osteodifferentiation, and yet in the absence of BMP-2, B2A is without cooperative effect.

Localization of colorectal carcinoma by rhenium-188-labeled B72.3 antibody in xenografted mice

In order to evaluate the feasibility of188Re-labeled antibodies for radioimmunotargeting, monoclonal antibody B72.3, recognizing TAG-72, expressed on the surface membranes of colorectal cancer cells, was directly labeled with188Re, obtained from a188W/188Re generator, using stannous tartrate and compared with125I-labeled B72.3. As a control, a human IgG was also radiolabeled with188Re andI25I. Prepared antibodies for188Re labeling could be stored as kits. Biodistribution was determined in nude mice inoculated with human colorectal carcinoma LoVo. Labeling efficiency and immunoreactivity of188Re-B72.3 were 80.3% and 64.7%, respectively.188Re-B72.3 localized specifically in the LoVo tumors. Although the absolute tumor accumulation level of188Re-B72.3 was lower thanI25I-B72.3,188Re-B72.3 demonstrated higher tumor-to-blood contrast than the125I-labeled counterpart, 2.04 ± 0.44 vs. 1.05 ± 0.28 at 96 hours, because of fast clearance from the blood.188Re-B72.3 seemed efficient for the imaging and therapy of colorectal carcinoma.

B2A, a receptor modulator, increases the growth of pluripotent and preosteoblast cells through bone morphogenetic protein receptors.

B2A (B2A2-K-NS) is a synthetic, multidomain peptide which is being developed to augment spinal fusion and bone repair locally. Using pluripotent mesenchymal cells of murine and human origin, we show that B2A-induced cell proliferation in a modest but dose-dependent manner. However, essentially all human tumor lines tested were not responsive or were weakly responsive to B2A. B2A treatment activated extracellular signal-regulated kinases 1 and 2 (ERK1/2), and the proliferation was partially blocked by an mitogen-activated protein kinase (MEK) inhibitor. The bone morphogenetic protein (BMP) type I receptor kinase inhibitors depressed B2A-induced proliferation. Upregulation of bone morphogenetic protein 2 was not involved, as noggin, DAN, or chordin did not block B2A-induced proliferation. These data suggest that B2A-induced proliferation results from cell-type-specific activation of bone morphogenetic protein receptor, which, in turn, regulates ERK1/2 activity. B2A-induced proliferation, acting through ERK1/2, is a phenomenon that, while not strictly related to the ability of B2A to augment BMP-induced differentiation via the small mothers against decapentaplegic pathway, may ultimately contribute to bone repair in vivo.

Similarity of copper and technetium binding sites in human IgG

Kits for direct labeling of IgG with 99mTc were used without modification for the preparation of [67Cu]IgG. The IgG was pre-treated to generate thiolate groups which would bind 67Cu. The direct labeling of reduced IgG with 67Cu was highly efficient, resulting in approx. 95% 67Cu binding. Non-reduced IgG (negative control) had labeling efficiencies of less than 10%. IgG pre-exposed to Cu(II) had reduced amounts of 99mTc bound to it. The results demonstrate a direct relationship between copper- and 99mTc-binding sites in IgG.