MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Tumor extracellular matrix has abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. In this work, we demonstrated effective MR cancer molecular imaging with a small molecular peptide targeted Gd-DOTA monoamide complex as a targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma. We performed the experiment of evaluating the effectiveness of the agent for non-invasive detection of prostate tumor with MRI in a mouse orthotopic PC-3 prostate cancer model. The targeted contrast agent was effective to produce significant tumor contrast enhancement at a low dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for MR molecular imaging of prostate tumor.     

转铁蛋白导向脂质体核磁造影剂纳米粒子（TfNIR-LipNBD-Magnevist）——一种肿瘤靶向磁共振造影剂

造影剂辅助的核磁共振成像是目前肿瘤诊断的最吁方法之一。但是由于核磁共振成像内在的低灵敏性以及造影剂的非特异性,导致肿瘤早期诊断较为困难。文章将一种新的肿瘤靶向核磁造影剂纳米粒子应用于早期肿瘤的影像诊断。这种新的肿瘤靶向核磁造影剂纳米粒子由配体转铁蛋白（Tf）、纳米水平的正电脂质体（Lip）载体和临床常用的造影剂Magnevist（Tf^NIR-Lip^NBD-Magnevist）三部分构成。另外转铁蛋白和脂质体粒子上,亦标记了荧光物质用于确定转铁蛋白一脂质体一造影剂纳米粒子的靶向性,以及肿瘤的光学影像诊断。在体外实验中,利用激光共聚焦显微镜和光学影像证明了靶向纳米粒子介导的细胞内吞和特异性结合。在裸鼠肿瘤模型中,造影剂纳米粒子Tf^NIR-Lip^NBD-Magnevist经尾静脉注入后,显著增强了肿瘤内信号与周围组织的对比度。由造影剂纳米粒子介导的肿瘤内信号显著强于单独Magnevist辅助的肿瘤内信号。同时,利用光学影像方法,在肿瘤内检测到特异的荧光信号。其结果进一步支持了转铁蛋白一脂质体一造影利（Tf^NIR-Lip^NBD-Magnevist）纳米粒子的靶向性和肿瘤影像诊断的有效性。     

Molecular magnetic resonance imaging with targeted contrast agents

Magnetic resonance imaging (MRI) produces high-resolution three-dimensional maps delineating morphological features of the specimen. Differential contrast in soft tissues depends on endogenous differences in water content, relaxation times, and/or diffusion characteristics of the tissue of interest. The specificity of MRI can be further increased by exogenous contrast agents (CA) such as gadolinium chelates, which have been successfully used for imaging of hemodynamic parameters including blood perfusion and vascular permeability. Development of targeted MR CA directed to specific molecular entities could dramatically expand the range of MR applications by combining the noninvasiveness and high spatial resolution of MRI with specific localization of molecular targets. However, due to the intrinsically low sensitivity of MRI (in comparison with nuclear imaging), high local concentrations of the CA at the target site are required to generate detectable MR contrast. To meet these requirements, the MR targeted CA should recognize targeted cells with high affinity and specificity. They should also be characterized by high relaxivity, which for a wide variety of CA depends on the number of contrast-generating groups per single molecule of the agent. We will review different designs and applications of targeted MR CA and will discuss feasibility of these approaches for in vivo MRI.     

Synthesis and Evaluation of a Peptide Targeted Small Molecular Gd-DOTA Monoamide Conjugate for MR Molecular Imaging of Prostate Cancer

Tumor extracellular matrix has an abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. Innovative design and development of safe and effective targeted contrast agents to these biomarkers would allow effective MR cancer molecular imaging with high spatial resolution. In this study, we synthesized a low molecular weight CLT1 peptide targeted Gd(III) chelate CLT1-dL-(Gd-DOTA)(4) specific to clotted plasma proteins in tumor stroma for cancer MR molecular imaging. CLT1-dL-(Gd-DOTA)(4) was synthesized by conjugating four Gd-DOTA monoamide chelates to a CLT1 peptide via generation 1 lysine dendrimer. The T(1) relaxivity of CLT1-dL-(Gd-DOTA)(4) was 40.4 mM(-1) s(-1) per molecule (10.1 mM(-1) s(-1) per Gd) at 37 °C and 1.5 T. Fluorescence imaging showed high binding specificity of CLT1 to orthotopic PC3 prostate tumor in mice. The contrast agent resulted in improved tumor contrast enhancement in male athymic nude mice bearing orthotopic PC3 prostate tumor xenograft at a dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for high-resolution MR molecular imaging of prostate tumor.     

Ultrasound Molecular Imaging of Secreted Frizzled Related Protein-2 Expression in Murine Angiosarcoma

Angiosarcoma is a biologically aggressive vascular malignancy with a high metastatic potential. In the era of targeted medicine, knowledge of specific molecular tumor characteristics has become more important. Molecular imaging using targeted ultrasound contrast agents can monitor tumor progression non-invasively. Secreted frizzled related protein 2 (SFRP2) is a tumor endothelial marker expressed in angiosarcoma. We hypothesize that SFRP2-directed imaging could be a novel approach to imaging the tumor vasculature. To develop an SFRP2 contrast agent, SFRP2 polyclonal antibody was biotinylated and incubated with streptavidin-coated microbubbles. SVR angiosarcoma cells were injected into nude mice, and when tumors were established the mice were injected intravenously with the SFRP2 -targeted contrast agent, or a control streptavidin-coated contrast agent. SFRP2 -targeted contrast agent detected tumor vasculature with significantly more signal intensity than control contrast agent: the normalized fold-change was 1.6±0.27 (n = 13, p = 0.0032). The kidney was largely devoid of echogenicity with no significant difference between the control contrast agent and the SFRP2-targeted contrast agent demonstrating that the SFRP2-targeted contrast agent was specific to tumor vessels. Plotting average pixel intensity obtained from SFRP2-targeted contrast agent against tumor volume showed that the average pixel intensity increased as tumor volume increased. In conclusion, molecularly-targeted imaging of SFRP2 visualizes angiosarcoma vessels, but not normal vessels, and intensity increases with tumor size. Molecular imaging of SFRP2 expression may provide a rapid, non-invasive method to monitor tumor regression during therapy for angiosarcoma and other SFRP2 expressing cancers, and contribute to our understanding of the biology of SFRP2 during tumor development and progression.     

Synthesis of tumor-targeted folate conjugated fluorescent magnetic albumin nanoparticles for enhanced intracellular dual-modal imaging into human brain tumor cells

Superparamagnetic iron oxide nanoparticles (SPIO NPs), utilized as carriers are attractive materials widely applied in biomedical fields, but target-specific SPIO NPs with lower toxicity and excellent biocompatibility are still lacking for intracellular visualization in human brain tumor diagnosis and therapy. Herein, bovine serum albumin (BSA) coated superparamagnetic iron oxide, i.e. γ-Fe₂O₃ nanoparticles (BSA-SPIO NPs), are synthesized. Tumor-specific ligand folic acid (FA) is then conjugated onto BSA-SPIO NPs to fabricate tumor-targeted NPs, FA-BSA-SPIO NPs as a contrast agent for MRI imaging. The FA-BSA-SPIO NPs are also labeled with fluorescein isothiocyanate (FITC) for intracellular visualization after cellular uptake and internalization by glioma U251 cells. The biological effects of the FA-BSA-SPIO NPs are investigated in human brain tumor U251 cells in detail. These results show that the prepared FA-BSA-SPIO NPs display undetectable cytotoxicity, excellent biocompatibility, and potent cellular uptake. Moreover, the study shows that the made FA-BSA-SPIO NPs are effectively internalized for MRI imaging and intracellular visualization after FITC labeling in the targeted U251 cells. Therefore, the present study demonstrates that the fabricated FITC-FA-BSA-SPIO NPs hold promising perspectives by providing a dual-modal imaging as non-toxic and target-specific vehicles in human brain tumor treatment in future.     

Molecular Magnetic Resonance Imaging of Tumors with a PTPµ Targeted Contrast Agent

Molecular magnetic resonance imaging (MRI) of tumors improves the specificity of MRI by using targeted probes conjugated to contrast-generating metals. The limitation of this approach is in the identification of a target molecule present in sufficient concentration for visualization and the development of a labeling reagent that can penetrate tumor tissue with the fast kinetics required for use in a clinical setting. The receptor protein tyrosine phosphatase PTPµ is a transmembrane protein that is continuously proteolyzed in the tumor microenvironment to generate a high concentration of extracellular fragment that can be recognized by the SBK2 probe. We conjugated the SBK2 peptide to a gadolinium chelate [SBK2-Tris-(Gd-DOTA)₃] to test whether the SBK2 probe could be developed as an MR molecular imaging probe. When intravenously injected into mice bearing flank tumors of human glioma cells, SBK2-Tris-(Gd-DOTA)₃ labeled the tumors within 5 minutes with a high level of contrast for up to 2 hours post-injection. The contrast enhancement of SBK2-Tris-(Gd-DOTA)₃ was significantly higher than that observed with a current MRI macrocyclic gadolinium chelate (Gadoteridol, ProHance) alone or a scrambled control. These results demonstrate that SBK2-Tris-(Gd-DOTA)₃ labeling of the PTPµ extracellular fragment is a more specific MR molecular imaging probe than ProHance or a scrambled control. Consequently, the SBK2 probe may be more useful than the current gold standard reagent for MRI to identify tumors and to co-register tumor borders during surgical resection.     

HER2 targeted molecular MR imaging using a de novo designed protein contrast agent

The application of magnetic resonance imaging (MRI) to non-invasively assess disease biomarkers has been hampered by the lack of desired contrast agents with high relaxivity, targeting capability, and optimized pharmacokinetics. We have developed a novel MR imaging probe targeting to HER2, a biomarker for various cancer types and a drug target for anti-cancer therapies. This multimodal HER20targeted MR imaging probe integrates a de novo designed protein contrast agent with a high affinity HER2 affibody and a near IR fluorescent dye. Our probe can differentially monitor tumors with different expression levels of HER2 in both human cell lines and xenograft mice models. In addition to its 100-fold higher dose efficiency compared to clinically approved non-targeting contrast agent DTPA, our developed agent also exhibits advantages in crossing the endothelial boundary, tissue distribution, and tumor tissue retention over reported contrast agents as demonstrated by even distribution of the imaging probe across the entire tumor mass. This contrast agent will provide a powerful tool for quantitative assessment of molecular markers, and improved resolution for diagnosis, prognosis and drug discovery.     

Development of contrast agents targeted to macrophage scavenger receptors for MRI of vascular inflammation

Atherosclerosis is a leading cause of death in the U.S. Because there is a potential to prevent coronary and arterial disease through early diagnosis, there is a need for methods to image arteries in the subclinical stage as well as clinical stage using various noninvasive techniques, including magnetic resonance imaging (MRI). We describe a development of a novel MRI contrast agent targeted to plaques that will allow imaging of lesion formation. The contrast agent is directed to macrophages, one of the earliest components of developing plaques. Macrophages are labeled through the macrophage scavenger receptor A, a macrophage specific cell surface protein, using an MRI contrast agent derived from scavenger receptor ligands. We have synthesized and characterized these contrast agents with a range of relaxivities. In vitro studies show that the targeted contrast agent accumulates in macrophages, and solution studies indicate that micromolar concentrations are sufficient to produce contrast in an MR image. Cell toxicity and initial biodistribution studies indicate low toxicity, no detectable retention in normal blood vessels, and rapid clearance from blood. The promising performance of this contrast agent targeted toward vascular inflammation opens doors to tracking of other inflammatory diseases such as tumor immunotherapy and transplant acceptance using MRI.     

A novel polyacrylamide magnetic nanoparticle contrast agent for molecular imaging using MRI

A novel polyacrylamide superparamagnetic iron oxide nanoparticle platform is described which has been synthetically prepared such that multiple crystals of iron oxide are encapsulated within a single polyacrylamide matrix (PolyAcrylamide Magnetic [PAM] nanoparticles). This formulation provides for an extremely large T2 and T2* relaxivity of between 620 and 1140 sec(-1) mM(-1). Administration of PAM nanoparticles into rats bearing orthotopic 9L gliomas allowed quantitative pharmacokinetic analysis of the uptake of nanoparticles in the vasculature, brain, and glioma. Addition of polyethylene glycol of varying sizes (0.6, 2, and 10 kDa) to the surface of the PAM nanoparticles resulted in an increase in plasma half-life and affected tumor uptake and retention of the nanoparticles as quantified by changes in tissue contrast using MRI. The flexible formulation of these nanoparticles suggests that future modifications could be accomplished allowing for their use as a targeted molecular imaging contrast agent and/or therapeutic platform for multiple indications.     

Selection of DNA Aptamers against Glioblastoma Cells with High Affinity and Specificity

Background

Glioblastoma is the most common and most lethal form of brain tumor in human. Unfortunately, there is still no effective therapy to this fatal disease and the median survival is generally less than one year from the time of diagnosis. Discovery of ligands that can bind specifically to this type of tumor cells will be of great significance to develop early molecular imaging, targeted delivery and guided surgery methods to battle this type of brain tumor.

Methodology/Principal Findings

We discovered two target-specific aptamers named GBM128 and GBM131 against cultured human glioblastoma cell line U118-MG after 30 rounds selection by a method called cell-based Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX). These two aptamers have high affinity and specificity against target glioblastoma cells. They neither recognize normal astraglial cells, nor do they recognize other normal and cancer cell lines tested. Clinical tissues were also tested and the results showed that these two aptamers can bind to different clinical glioma tissues but not normal brain tissues. More importantly, binding affinity and selectivity of these two aptamers were retained in complicated biological environment.

Conclusion/Significance

The selected aptamers could be used to identify specific glioblastoma biomarkers. Methods of molecular imaging, targeted drug delivery, ligand guided surgery can be further developed based on these ligands for early detection, targeted therapy, and guided surgery of glioblastoma leading to effective treatment of glioblastoma.     

Chlorophyll-a analogues conjugated with aminobenzyl-DTPA as potential bifunctional agents for magnetic resonance imaging and photodynamic therapy

A clinically relevant photosensitizer, 3-devinyl-3-(1-hexyloxyethyl)pyropheophorbide-a (HPPH, a chlorophyll-a derivative), was conjugated with Gd(III)-aminobenzyl-diethylenetriaminepentaacetic acid (DTPA), an experimental magnetic resonance (MR) imaging agent. In vivo reflectance spectroscopy confirmed tumor uptake of HPPH-aminobenzyl-Gd(III)-DTPA conjugate was higher than free HPPH administered intraveneously (iv) to C3H mice with subcutaneously (sc) implanted radiation-induced fibrosarcoma (RIF) tumor cells. In other experiments, Sprague-Dawley (SD) rats with sc implanted Ward Colon Carcinoma cells yielded markedly increased MR signal intensities from tumor regions-of-interest (ROIs) 24 h post-iv injection of HPPH-aminobenzyl-Gd(III)-DTPA conjugate as compared to unconjugated HPPH. In both in vitro (RIF tumor cells) and in vivo (mice bearing RIF tumors and rats bearing Ward Colon tumors) the conjugate produced significant increases in tumor conspicuity at 1.5 T and retained therapeutic efficacy following PDT. Also synthesized were a series of novel bifunctional agents containing two Gd(III) atoms per HPPH molecule that remained tumor-avid and PDT-active and yielded improved MR tumor conspicuity compared to their corresponding mono-Gd(III) analogues. Administered iv at a MR imaging dose of 10 micromol/kg, these conjugates produced severe skin phototoxicity. However, by replacing the hexyl group of the pyropheophorbide-a with a tri(ethylene glycol) monomethyl ether (PEG-methyl ether), these conjugates produced remarkable MR tumor enhancement at 8 h post-iv injection, significant tumoricidal activity (80% of mice were tumor-free on day 90), and reduced skin phototoxicity compared to their corresponding hexyl ether analogues. The poor water-solubility characteristic of these conjugates was resolved by incorporation into a liposomal formulation. This paper presents the synthesis of tumor-avid contrast enhancing agents for MR imaging and thus represents an important milestone toward improving cancer diagnosis and tumor characterization. More importantly, this paper describes a new family of bifunctional agents that combine two modalities into a single cost-effective "see and treat" approach, namely, a single agent that can be used for contrast agent-enhanced MR imaging followed by targeted photodynamic therapy.     

Specific Visualization of Glioma Cells in Living Low-Grade Tumor Tissue

Background

The current therapy of malignant gliomas is based on surgical resection, radio-chemotherapy and chemotherapy. Recent retrospective case-series have highlighted the significance of the extent of resection as a prognostic factor predicting the course of the disease. Complete resection in low-grade gliomas that show no MRI-enhanced images are especially difficult. The aim in this study was to develop a robust, specific, new fluorescent probe for glioma cells that is easy to apply to live tumor biopsies and could identify tumor cells from normal brain cells at all levels of magnification.

Methodology/Principal Findings

In this investigation we employed brightly fluorescent, photostable quantum dots (QDs) to specifically target epidermal growth factor receptor (EGFR) that is upregulated in many gliomas. Living glioma and normal cells or tissue biopsies were incubated with QDs coupled to EGF and/or monoclonal antibodies against EGFR for 30 minutes, washed and imaged. The data include results from cell-culture, animal model and ex vivo human tumor biopsies of both low-grade and high-grade gliomas and show high probe specificity. Tumor cells could be visualized from the macroscopic to single cell level with contrast ratios as high as 1000: 1 compared to normal brain tissue.

Conclusions/Significance

The ability of the targeted probes to clearly distinguish tumor cells in low-grade tumor biopsies, where no enhanced MRI image was obtained, demonstrates the great potential of the method. We propose that future application of specifically targeted fluorescent particles during surgery could allow intraoperative guidance for the removal of residual tumor cells from the resection cavity and thus increase patient survival.     

A dual function fusion protein of Herpes simplex virus type 1 thymidine kinase and firefly luciferase for noninvasive in vivo imaging of gene therapy in malignant glioma

BACKGROUND: Suicide gene therapy employing the prodrug activating system Herpes simplex virus type 1 thymidine kinase (HSV-TK)/ ganciclovir (GCV) has proven to be effective in killing experimental brain tumors. In contrast, glioma patients treated with HSV-TK/ GCV did not show significant treatment benefit, most likely due to insufficient transgene delivery to tumor cells. Therefore, this study aimed at developing a strategy for real-time noninvasive in vivo monitoring of the activity of a therapeutic gene in brain tumor cells. METHODS: The HSV-TK gene was fused to the firefly luciferase (Luc) gene and the fusion construct HSV-TK-Luc was expressed in U87MG human malignant glioma cells. Nude mice with subcutaneous gliomas stably expressing HSV-TK-Luc were subjected to GCV treatment and tumor response to therapy was monitored in vivo by serial bioluminescence imaging. Bioluminescent signals over time were compared with tumor volumes determined by caliper. RESULTS: Transient and stable expression of the HSV-TK-Luc fusion protein in U87MG glioma cells demonstrated close correlation of both enzyme activities. Serial optical imaging of tumor bearing mice detected in all cases GCV induced death of tumor cells expressing the fusion protein and proved that bioluminescence can be reliably used for repetitive and noninvasive quantification of HSV-TK/ GCV mediated cell kill in vivo. CONCLUSION: This approach may represent a valuable tool for the in vivo evaluation of gene therapy strategies for treatment of malignant disease.     

Pyropheophorbide 2-deoxyglucosamide: a new photosensitizer targeting glucose transporters

To prepare near-infrared fluorescence imaging and photodynamic therapy agents targeted at glucose transporters, pyropheophorbide 2-deoxyglucosamide (Pyro-2DG) was synthesized and evaluated in a 9L glioma rat model. Fluorescence imaging studies demonstrate that Pyro-2DG is selectively accumulated in the tumor. Upon its photoactivation, we demonstrate that this agent efficiently causes selective mitochondrial damage to the region of a tumor that was photoirradiated after administration of this agent, but does not affect tissues photoirradiated in the absence of the agent or tissues treated with the agent that are not photoirradiated. Preliminary confocal microscopy studies suggest that Pyro-2DG is delivered and trapped in tumor cells via the GLUT/hexokinase pathway and therefore is useful both as a tumor-targeted NIR fluorescence imaging probe and as a PDT agent for the destruction of cancer.     

Phenyl-tert-butylnitrone induces tumor regression and decreases angiogenesis in a C6 rat glioma model

The prognosis of patients who are diagnosed with glioblastoma multiforme is very poor, due to the difficulty of an early and accurate diagnosis and the lack of currently efficient therapeutic compounds. The efficacy of phenyl-tert-butylnitrone (PBN) as a potential anti-glioma therapeutic drug was assessed by magnetic resonance (MR) imaging (T(1)/T(2)-weighted imaging) and MR angiography (time-of-flight imaging, in conjunction with a Mathematica-based program) methods by monitoring morphologic properties, growth patterns, and angiogenic behaviors of a moderately aggressive rat C6 glioma model. MR results from untreated rats showed the diffusive invasiveness of C6 gliomas, with some associated angiogenesis. PBN administration as a pretreatment was found to clearly induce a decrease in growth rate and tumor regression as well as preventing angiogenesis. This compound even had a 40% efficiency in reducing well-established tumors. MR findings rivaled those from histology and angiogenesis marker immunostaining evaluations. In this study we demonstrated the efficiency of PBN as a potential anti-glioma drug and found it to inhibit tumor cell proliferation and prevent vascular alterations in early stages of glioma progression. The MR methods that we used also proved to be particularly suitable in following the angiogenic behavior and treatment response of a potential anti-glioma agent in a rat C6 glioma model.     

Targeted gadolinium-loaded dendrimer nanoparticles for tumor-specific magnetic resonance contrast enhancement

A target-specific MRI contrast agent for tumor cells expressing high affinity folate receptor was synthesized using generation five (G5) ofpolyamidoamine (PAMAM) dendrimer. Surface modified dendrimer was functionalized for targeting with folic acid (FA) and the remaining terminal primary amines of the dendrimer were conjugated with the bifunctional NCS-DOTA chelator that forms stable complexes with gadolinium (Gd III). Dendrimer-DOTA conjugates were then complexed with GdCl3 followed by ICP-OES as well as MRI measurement of their longitudinal relaxivity (T1 s(-1) mM(-1)) of water. In xenograft tumors established in immunodeficient (SCID) mice with KB human epithelial cancer cells expressing folate receptor (FAR), the 3D MRI results showed specific and statistically significant signal enhancement in tumors generated with targeted Gd(III)-DOTA-G5-FA compared with signal generated by non-targeted Gd(III)-DOTA-G5 contrast nanoparticle. The targeted dendrimer contrast nanoparticles infiltrated tumor and were retained in tumor cells up to 48 hours post-injection of targeted contrast nanoparticle. The presence of folic acid on the dendrimer resulted in specific delivery of the nanoparticle to tissues and xenograft tumor cells expressing folate receptor in vivo. We present the specificity of the dendrimer nanoparticles for targeted cancer imaging with the prolonged clearance time compared with the current clinically approved gadodiamide (Omniscan) contrast agent. Potential application of this approach may include determination of the folate receptor status of tumors and monitoring of drug therapy.     

Efficient and selective gene transfer into primary human brain tumors by using single-chain antibody-targeted adenoviral vectors with native tropism abolished

The application of adenoviral vectors in cancer gene therapy is hampered by low receptor expression on tumor cells and high receptor expression on normal epithelial cells. Targeting adenoviral vectors toward tumor cells may improve cancer gene therapy procedures by providing augmented tumor transduction and decreased toxicity to normal tissues. Targeting requires both the complete abolition of native tropism and the addition of a new specific binding ligand onto the viral capsid. Here we accomplished this by using doubly ablated adenoviral vectors, lacking coxsackievirus-adenovirus receptor and alpha(v) integrin binding capacities, together with bispecific single-chain antibodies targeted toward human epidermal growth factor receptor (EGFR) or the epithelial cell adhesion molecule. These vectors efficiently and selectively targeted both alternative receptors on the surface of human cancer cells. Targeted doubly ablated adenoviral vectors were also very efficient and specific with primary human tumor specimens. With primary glioma cell cultures, EGFR targeting augmented the median gene transfer efficiency of doubly ablated adenoviral vectors 123-fold. Moreover, EGFR-targeted doubly ablated vectors were selective for human brain tumors versus the surrounding normal brain tissue. They transduced organotypic glioma and meningioma spheroids with efficiencies similar to those of native adenoviral vectors, while exhibiting greater-than-10-fold-reduced background levels on normal brain explants from the same patients. As a result, EGFR-targeted doubly ablated adenoviral vectors had a 5- to 38-fold-improved tumor-to-normal brain targeting index compared to native vectors. Hence, single-chain targeted doubly ablated adenoviral vectors are promising tools for cancer gene therapy. They should provide an improved therapeutic index with efficient tumor transduction and effective protection of normal tissue.     

Development of SM5-1-conjugated ultrasmall superparamagnetic iron oxide nanoparticles for hepatoma detection

Ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles are maghemite or magnetite nanoparticles currently used as contrast agent in magnetic resonance imaging (MRI). In this study, a targeted contrast agent (SM-USPIO) was prepared by conjugating coprecipitated USPIO to a humanized SM5-1 antibody which can specifically react with human hepatocellular carcinoma (HCC) cells. The binding and internalization of SM-USPIO to the HCC cell line ch-hep-3 was confirmed by flow cytometry and confocal microscopy. Furthermore, SM-USPIO was demonstrated to be able to selectively accumulate in the tumor cells, resulting in a marked decrease of MRI T2-weighted signal intensity. Biodistribution studies demonstrated the efficient accumulation of SM-USPIO in the ch-hep-3 tumor in nude mice. The in vivo study in the ch-hep-3 tumor-bearing nude mice indicated that MRI using the SM-USPIO as contrast agent possessed good diagnostic ability, suggesting that SM-USPIO had the potential to be a promising targeted contrast agent for diagnosis of HCC.     

Polycefin, a new prototype of a multifunctional nanoconjugate based on poly(beta-L-malic acid) for drug delivery

A new prototype of nanoconjugate, Polycefin, was synthesized for targeted delivery of antisense oligonucleotides and monoclonal antibodies to brain tumors. The macromolecular carrier contains: 1. biodegradable, nonimmunogenic, nontoxic beta-poly(L-malic acid) of microbial origin; 2. Morpholino antisense oligonucleotides targeting laminin alpha4 and beta1 chains of laminin-8, which is specifically overexpressed in glial brain tumors; 3. monoclonal anti-transferrin receptor antibody for specific tissue targeting; 4. oligonucleotide releasing disulfide units; 5. L-valine containing, pH-sensitive membrane disrupting unit(s), 6. protective poly(ethylene glycol); 7. a fluorescent dye (optional). Highly purified modules were conjugated directly with N-hydroxysuccinimidyl ester-activated beta-poly(L-malic acid) at pendant carboxyl groups or at thiol containing spacers via thioether and disulfide bonds. Products were chemically validated by physical, chemical, and functional tests. In vitro experiments using two human glioma cell lines U87MG and T98G demonstrated that Polycefin was delivered into the tumor cells by a receptor-mediated endocytosis mechanism and was able to inhibit the synthesis of laminin-8 alpha4 and beta1 chains at the same time. Inhibition of laminin-8 expression was in agreement with the designed endosomal membrane disruption and drug releasing activity. In vivo imaging showed the accumulation of intravenously injected Polycefin in brain tumor tissue via the antibody-targeted transferrin receptor-mediated endosomal pathway in addition to a less efficient mechanism known for high molecular mass biopolymers as enhanced permeability and retention effect. Polycefin was nontoxic to normal and tumor astrocytes in a wide range of concentrations, accumulated in brain tumor, and could be used for specific targeting of several biomarkers simultaneously.