Polymeric 1 H MRI Probes for Visualizing Tumor In Vivo.

Magnetic resonance imaging (MRI) has become a prominent non- or low-invasive imaging technique, providing high-resolution, three-dimensional images as well as physiological information about tissues. Low-molecular-weight Gd-MRI contrast agents (CAs), such as Gd-DTPA (DTPA: diethylenetriaminepentaacetic acid), are commonly used in the clinical diagnosis, while macromolecular Gd-MRI CAs have several advantages over low-molecular-weight Gd-MRI CAs, which help minimize the dose of CAs and the risk of side effects. Accordingly, we developed chiral dendrimer Gd-MRI CAs, which showed high r1 values. The association constant values (Ka ) of S-isomeric dendrimer CAs to bovine serum albumin (BSA) were higher than those of R-isomeric dendrimer CAs. Besides, based on a totally new concept, we developed 13 C/15 N-enriched multiple-resonance NMR/MRI probes, which realized highly selective observation of the probes and analysis of metabolic reactions of interest. This account summarizes our recent study on developing both chiral dendrimer Gd-MRI CAs, and self-traceable 13 C/15 N-enriched phosphorylcholine polymer probes for early detection of tumors.

Magnetic resonance imaging of tumor with a self-traceable phosphorylcholine polymer.

Polymers are concentration-amplified with respect to the monomeric units. We show here that a phosphorylcholine polymer enriched with (13)C/(15)N at the methyl groups is self-traceable by multiple-resonance (heteronuclear-correlation) NMR in tumor-bearing mice inoculated with the mouse rectal cancer cell line (colon 26). Preliminary measurements indicated that the present polymeric nanoprobe was satisfactorily distinguished from lipids and detectable with far sub-micromolar spectroscopic and far sub-millimolar imaging sensitivities. Detailed ex vivo and in vivo studies for the tumor-bearing mice administered the probe with a mean molecular weight of 63,000 and a mean size of 13 nm, revealed the following: (1) this probe accumulates in the tumor highly selectively (besides renal excretion) and efficiently (up to 30% of the injected dose), (2) the tumor can thus be clearly in vivo imaged, the lowest clearly imageable dose of the probe being 100 mg/kg or 2.0 mg/20-g mouse, and (3) the competition between renal excretion and tumor accumulation is size-controlled; that is, the larger (higher molecular-weight) and smaller (lower molecular-weight) portions of the probe undergo tumor accumulation and renal excretion, respectively. The observed size dependence suggests that the efficient tumor-targeting of the present probe is stimulated primarily by the so-called enhanced permeability and retention (EPR) effect, that is, size-allowed invasion of the probe into the tumor tissue via defective vascular wall. Self-traceable polymers thus open an important area of magnetic resonance imaging (MRI) of tumors and may provide a highly potential tool to visualize various delivery/localization processes using synthetic polymers.

Magnetic resonance imaging of tumor with a self-traceable polymer conjugated with an antibody fragment.

A (13)C-enriched phosphorylcholine polymer ((13)C-PMPC) as a self-traceable MR (magnetic resonance) tag was conjugated with a fragment (scFv) of Herceptin, a clinical antibody against antigen Her2. When injected in model mice bearing Her2(+) (gastric) and Her2(-) (pancreatic) tumors, the antibody-tag conjugate (13)C-PMPC-scFv selectively accumulated in the Her2(+) tumor with a rapid build-up/decay (accumulation/clearance) profile and, with the use of the (1)H-(13)C double-resonance (heteronuclear correlation) technique, the Her2(+) gastric tumor was clearly MR imaged.

A Comparative Study of Tumor-Specificity and Neurotoxicity between 3-Styrylchromones and Anti-Cancer Drugs.

Background. Many anti-cancer drugs used in clinical practice cause adverse events such as oral mucositis, neurotoxicity, and extravascular leakage. We have reported that two 3-styrylchromone derivatives, 7-methoxy-3-[(1E)-2-phenylethenyl]-4H-1-benzopyran-4-one (Compound A) and 3-[(1E)-2-(4-hydroxyphenyl)ethenyl]-7-methoxy-4H-1-benzopyran-4-one (Compound B), showed the highest tumor-specificity against human oral squamous cell carcinoma (OSCC) cell lines among 291 related compounds. After confirming their superiority by comparing their tumor specificity with newly synthesized 65 derivatives, we investigated the neurotoxicity of these compounds in comparison with four popular anti-cancer drugs. Methods: Tumor-specificity (TSM, TSE, TSN) was evaluated as the ratio of mean CC50 for human normal oral mesenchymal (gingival fibroblast, pulp cell), oral epithelial cells (gingival epithelial progenitor), and neuronal cells (PC-12, SH-SY5Y, LY-PPB6, differentiated PC-12) to OSCC cells (Ca9-22, HSC-2), respectively. Results: Compounds A and B showed one order of magnitude higher TSM than newly synthesized derivatives, confirming its prominent tumor-specificity. Docetaxel showed one order of magnitude higher TSM, but two orders of magnitude lower TSE than Compounds A and B. Compounds A and B showed higher TSM, TSE, and TSN values than doxorubicin, 5-FU, and cisplatin, damaging OSCC cells at concentrations that do not affect the viability of normal epithelial and neuronal cells. QSAR prediction based on the Tox21 database suggested that Compounds A and B may inhibit the signaling pathway of estrogen-related receptors.

Human placental ectonucleoside triphosphate diphosphohydrolase gene transfer via gelatin-coated stents prevents in-stent thrombosis.

BACKGROUND: In-stent thrombosis is mainly triggered by adenosine diphosphate (ADP)-dependent platelet aggregation after percutaneous coronary stent implantation. Ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) rapidly hydrolyzes ADP to adenosine monophosphate, inhibiting platelet aggregation. We tested the hypothesis that local delivery of human placental E-NTPDase (pE-NTPDase) gene into injured arteries via gene-eluting stent could prevent subacute in-stent thrombosis. METHODS AND RESULTS: We generated gene-eluting stents by coating bare metal stents with cationic gelatin hydrogel containing pE-NTPDase cDNA (pE-NTPDase stent), and implanted the stents into rabbit femoral arteries (FA) prone to production of platelet-rich thrombi due to repeated balloon injury at 4-week intervals. After the second injury, E-NTPDase gene expression was severely decreased; however, the implantation of pE-NTPDase stent increased E-NTPDase mRNA levels and NTPDase activity to higher level than normal FA. The FAs with pE-NTPDase stents maintained patency in all rabbits (P<0.01), whereas the stent-implanted FAs without pE-NTPDase gene showed low patency rates (17% to 25%). The occlusive platelet-rich thrombi, excessive neointimal growth, and infiltration of macrophages were inhibited in stent implanted FA with pE-NTPDase gene, but not without pE-NTPDase gene. CONCLUSIONS: Human pE-NTPDase gene transfer via cationic gelatin-coated stents inhibited subacute in-stent thrombosis and suppressed neointimal hyperplasia and inflammation without antiplatelet drugs.

Photoacoustic in vivo 3D imaging of tumor using a highly tumor-targeting probe under high-threshold conditions.

Three-dimensional (3D) representation of a tumor with respect to its size, shape, location, and boundaries is still a challenge in photoacoustic (PA) imaging using artificial contrast agents as probes. We carried out PA imaging of tumors in mice using 800RS-PMPC, which was obtained by coupling of 800RS, a near-infrared cyanine dye, with PMPC, a highly selective tumor-targeting methacrylate polymer having phosphorylcholine side chains, as a probe. The conjugate 800RS-PMPC forms compact nanoparticles (dDLS = 14.3 nm), retains the biocompatibility of the parent polymer (PMPC) and exhibits unprecedented PA performance. When applied to mice bearing a 6 × 3 × 3 mm3 tumor buried 6 mm beneath the skin, the probe 800RS-PMPC selectively accumulates in the tumor and emits PA signals that are strong enough to be unambiguously distinguished from noise signals of endogenous blood/hemoglobin. The PA image thus obtained under high-threshold conditions allows 3D characterization of the tumor in terms of its size, shape, location, and boundaries.

Experimental tissue regeneration by DDS technology of bio-signaling molecules.

The medical therapy of tissue regeneration achieved by biomaterial-based tissue engineering has been currently expected as the third option following reconstructive surgery and organ transplantation. The basic idea of this regenerative therapy is to assist the self-healing potentials of body to induce the natural regeneration and repairing of defective or injured tissue. To this end, it is practically important to create a local environment which enables cells to promote their proliferation and differentiation, resulting in the induction of cell-based tissue regeneration. Tissue engineering is a biomedical technology or methodology to build up this regeneration environment by making use of biomaterials. Drug delivery system (DDS) is a biomaterial technology to enhance the in vivo biological functions of bio-signaling molecules (growth factors and genes) for promoted tissue regeneration. This paper overviews the recent status of tissue regeneration therapy based on the DDS technology of bio-signaling molecules.

In vivo release of plasmid DNA from composites of oligo(poly(ethylene glycol)fumarate) and cationized gelatin microspheres.

Composites of cationized gelatin microspheres (CGMS), crosslinked with either 3 mM or 6 mM glutaraldehyde solution, and a novel hydrogel material, oligo(poly(ethylene glycol)fumarate) (OPF) were fabricated and investigated toward prolonging the release of plasmid DNA in vivo relative to the constituent materials. The composites and constituent materials were investigated in a subcutaneous murine model to assess the release of 125I-labeled plasmid DNA and 125I-labeled cationized gelatin in vivo. The time profiles of the radioactivity remaining were employed to compare the profiles of DNA release and cationized gelatin degradation. Both composite formulations (incorporating either 3 mM or 6 mM CGMS) prolonged the bioavailability of plasmid DNA relative to both injected plasmid DNA solution and the respective non-embedded cationized gelatin microspheres. Injected plasmid DNA solution persisted in the subject for only 7-10 days, whereas the persistence of DNA from composites of OPF and either 3 mM or 6 mM CGMS extended to at least day 42. The 3 mM and 6 mM CGMS each increased the persistence of DNA slightly, relative to injection of DNA solution, to between 28 and 35 days. Interestingly, the release profile of plasmid DNA from composites was not significantly different from the release of DNA from OPF alone. The release of plasmid DNA from the composites was in accord with the degradation of the microspheres within the OPF. These results show that composites of OPF and cationized gelatin microspheres are able to prolong the availability of plasmid DNA in vivo relative to cationized gelatin microspheres alone and provide a promising candidate material for the sustained, controlled release of plasmid DNA.

Fabrication and biocompatibility of collagen sponge reinforced with poly(glycolic acid) fiber.

This article describes an investigation of collagen sponge mechanically reinforced through the incorporation of poly(glycolic acid) (PGA) fiber. A collagen solution with PGA fiber homogeneously dispersed at collagen:PGA weight ratios of 1.5, 0.8, 0.4, and 0.2 was freeze-dried, followed by dehydrothermal cross-linking to obtain collagen sponges incorporating PGA fiber to various extents. By scanning electron microscopy observation, the collagen sponges exhibited isotropic and interconnected pore structures with an average size of 180 microm, irrespective of PGA fiber incorporation. As expected, PGA fiber incorporation enabled the collagen sponges to significantly enhance their compression strength. In vitro cell culture studies revealed that the number of L929 fibroblasts initially attached was significantly greater for any collagen sponge incorporating PGA fiber than for collagen sponge. The shrinkage of sponge after cell seeding was suppressed by fiber incorporation. It is possible that shrinkage suppression results in the superior cell attachment of sponge incorporating PGA fiber. After subcutaneous implantation into the backs of mice, the residual volume of collagen sponge incorporating PGA fiber was significant compared with that of collagen sponge and increased with a decrease in the collagen:PGA ratio. The greater number of cells infiltrated and deeper infiltration were observed for collagen sponge incorporating PGA fiber implanted subcutaneously. We conclude that the incorporation of PGA fiber is a simple and promising way to reinforce collagen sponge without impairing biocompatibility.

Size-controlled and biocompatible Gd2 O3 nanoparticles for dual photoacoustic and MR imaging.

The synthesis, characterization, and functional evaluation of new size-controlled and biocompatible Gd(2) O(3) nanoparticles as a bimodal contrast agent for use in photoacoustic (PA) and magnetic resonance (MR) imaging are reported. These nanoparticles show a clear PA image by themselves, without conjugation with gold, rare earth metals, or dyes. Relaxivity measurement by MR imaging clearly shows that their relaxivity, r(1) , is twice that of clinically available Gd-DTPA.

Sustained bFGF-release tubes for peripheral nerve regeneration: comparison with autograft.

BACKGROUND: Despite numerous articles on the use of artificial nerve conduits, autologous nerve transplants remain the most effective for nerve repair. To improve this technique, the authors examined conduits containing gelatin hydrogel as a carrier enabling the sustained release of basic fibroblast growth factor (bFGF). METHODS: To confirm sustained bFGF release in vivo, nerve-guide tubes containing iodine-125-labeled bFGF with or without gelatin hydrogel were implanted under the skin of mice, and the remaining radioactivity was measured. Next, a 15-mm segment of the sciatic nerve was resected and repaired with autologous nerve (group 1), a tube with gelatin hydrogel and bFGF (group 2), a tube with bFGF alone (group 3), or a tube only (group 4). Histologic and functional analyses were performed for 16 weeks after surgery. RESULTS: The radioactivity from iodine-125-labeled bFGF incorporated into gelatin hydrogel decreased more slowly than iodine-125-labeled bFGF alone. Four weeks after surgery, significantly more regenerating axons were detected in group 2 than in groups 3 and 4, but the axonal density in group 2 was lower than in group 1. Similarly, the animals in group 2 showed significantly better motor performance than those in groups 3 and 4, but worse than those in group 1. The animals in groups 1 and 2 showed significantly better sensory recovery than those in groups 3 and 4. CONCLUSIONS: The nerve-guide tube containing gelatin hydrogel and bFGF promoted axonal regeneration after peripheral nerve injury, but not as well as autologous transplants. Understanding the limitations of this technique will facilitate its improvement for clinical applications.

A novel gelatin hydrogel carrier sheet for corneal endothelial transplantation.

We examined the feasibility of using gelatin hydrogels as carrier sheets for the transplantation of cultivated corneal endothelial cells. The mechanical properties, transparency, and permeability of gelatin hydrogel sheets were compared with those of atelocollagen sheets. Immunohistochemistry (ZO-1, Na(+)/K(+)-ATPase, and N-cadherin), hematoxylin and eosin staining, and scanning electron microscopy were performed to assess the integrity of corneal endothelial cells that were cultured on gelatin hydrogel sheets. The gelatin hydrogel sheets displayed greater transparency, elastic modulus, and albumin permeability compared to those of atelocollagen sheets. The corneal endothelial cells on gelatin hydrogel sheets showed normal expression levels of ZO-1, Na(+)/K(+)-ATPase, and N-cadherin. Hematoxylin and eosin staining revealed the formation of a continuous monolayer of cells attached to the gelatin hydrogel sheet. Scanning electron microscopy observations showed that the corneal endothelial cells were arranged in a regular, mosaic, and polygonal pattern with normal cilia. These results indicate that the gelatin hydrogel sheet is a promising material to transport corneal endothelial cells during transplantation.

Effect of gelatin hydrogel incorporating fibroblast growth factor 2 on human meniscal cells in an organ culture model.

Efforts to use growth factors to enhance the healing potential of the meniscus have been impeded because their half-lives are too short to maintain the biological activity. The thread was coated with gelatin hydrogel and fibroblast growth factor 2 (FGF 2) was biologically stabilized by incorporating in a gelatin hydrogel-coated thread. The purpose of this study is to investigate the effect of gelatin hydrogel-coated thread incorporating FGF 2 on human meniscal cells in an organ culture. Twenty-five menisci were cut into small pieces, and selected pieces were sutured with gelatin hydrogel-coated thread incorporating FGF 2 (FGF(+) group) or physiologic saline (FGF(-) group), followed by organ culture. The meniscal samples histologically evaluated 4, 7, and 14 days later. The cell density and the number of PCNA-positive cells for the FGF(+) group were higher than those of the FGF(-) group, while the number of TUNEL-positive cells was lower. These results suggest that FGF 2 stimulates the proliferation of meniscal cells and inhibits meniscal cell death. Gelatin hydrogel-coated threads releasable FGF 2 may be useful to promote repairing of human meniscus.

Preparation of collagen/gelatin sponge scaffold for sustained release of bFGF.

Artificial dermis (AD) has been used to regenerate dermis-like tissues in the treatment of full-thickness skin defects, but it takes 2 or 3 weeks to complete dermal regeneration. Our previous study demonstrated that injection of basic fibroblast growth factor (bFGF)-impregnated gelatin microspheres (MS) into the AD accelerates the regeneration of dermis-like tissue. However, injection of gelatin MS before clinical use is complicated and time consuming. This study investigated a new scaffold, in which collagen and gelatin are integrated, and which is capable of sustained bFGF release. We produced collagen/gelatin sponges with a gelatin concentration of 0wt%, 10wt%, 30wt%, and 50wt%. The mean pore size in each sponge decreased with the gelatin concentration. In an in vitro study, proliferation of fibroblasts in each sponge was not significantly different over 7 days of culture. As for in vivo sustained release of bFGF, a radioisotope study demonstrated that retention of bFGF in gelatin 10wt% and 30wt% sponges was significantly larger than that in gelatin 0wt% sponge. The collagen/gelatin sponges were grafted on full-thickness skin defects created on a rabbit ear, and we evaluated regeneration of dermis-like tissue by measuring the amount of hemoglobin and size of dermis-like tissue on histological sections. Seven days after implantation, the amount of hemoglobin in dermis-like tissue in gelatin 10wt% sponge was significantly larger than those in control and gelatin 50wt% sponge. Twenty-eight days after implantation, the area of dermis-like tissue in gelatin 10wt% sponge was significantly larger than those in the other specimens. We conclude that the collagen sponge integrated with 10wt% gelatin has the most potential for sustained release of bFGF and that the combination of collagen/gelatin 10wt% sponge and bFGF is a promising therapeutic modality for the treatment of full-thickness skin defects.

Hydrogel-mediated release of basic fibroblast growth factor from a stent-graft accelerates biological fixation with the aortic wall in a porcine model.

PURPOSE: To evaluate the local reaction of the aortic wall induced by basic fibroblast growth factor (bFGF) released from a gelatin hydrogel coated on the outer surface of a stent-graft for the purpose of biological fixation. METHODS: A total of 18 nitinol-based, polyester-covered stent-grafts were implanted in 6 porcine aortas for 1 month. The implanted stent-grafts were divided into 3 groups: the control group (uncoated), the hydrogel group (coated with hydrogel containing water), and the bFGF group (coated with hydrogel containing bFGF). After stent-graft implantation, the results of intravascular ultrasound (IVUS) and qualitative and quantitative microscopic examinations were compared among the groups. RESULTS: In the bFGF group, a thin white lamellar tissue was observed on IVUS images. Significantly more new intimal tissue formation was observed in all the bFGF group animals than in the other 2 groups, and alpha smooth muscle (SM) actin-positive cells (alphaSMCs) were detected in this new tissue. The alphaSMCs within the fabric of tightly woven grafts were significantly more abundant in the bFGF group than in the other groups. CONCLUSION: The local controlled release of bFGF from the stent-graft significantly accelerated the proliferation of new intimal tissue between the aorta and the stent-graft and within the graft materials. These findings suggest that a graft can be fixed biologically to the aortic wall, which may contribute to the shrinkage of aneurysms following stent-grafting.

A novel approach to reduce catheter-related infection using sustained-release basic fibroblast growth factor for tissue regeneration in mice.

Catheter-related infection is one of the most serious complications. Microbes migrate along the catheter (the foreign material) from the wound at the insertion-site, leading to catheter-related infection. Basic fibroblast growth factor (bFGF) is a potent mitogen that promotes the growth and regeneration of organs and tissues in vivo. Catheter-related bacterial invasion was simulated by the invasion of inoculated bacteria into a transplanted foreign material. Sterile Dacron sheets (foreign materials) were implanted on the subcutis of 96 male mice (C57BL/6) randomized into four groups (n = 24 per group). Group A: Dacron sheets only; Group B: Dacron sheets treated with a plain gelatin hydrogel sheet; Group C: Dacron sheets treated with free bFGF (50 microg); Group D: Dacron sheets treated with sustained-release bFGF (50 microg). On day 7, "detachment test" (to measure the force needed to pull out the Dacron sheet) and microscopic evaluations were performed, and the tissue immediately above the Dacron sheet was inoculated with methicillin-resistant Staphylococcus aureus (MRSA) 1 x 10(6) colony-forming units. The total energy needed for pulling out the implanted Dacron sheet in Group D was significantly higher than other three groups (P < 0.01). Group D had a large granulation tissue area containing a large amount of collagen tissue and vessels microscopically. Two days after the MRSA inoculation, the number of MRSA in the Dacron sheet of Group D was smallest. Pretreatment with sustained-release form of bFGF promoted tissue regeneration and reduced catheter-related bacterial invasion, indicating a useful adjuvant for reducing catheter-related infection.