Debadyuti Ghosh

M13-templated magnetic nanoparticles for targeted in vivo imaging of prostate cancer

Molecular imaging allows clinicians to visualize the progression of tumours and obtain relevant information for patient diagnosis and treatment. Owing to their intrinsic optical, electrical and magnetic properties, nanoparticles are promising contrast agents for imaging dynamic molecular and cellular processes such as protein-protein interactions, enzyme activity or gene expression. Until now, nanoparticles have been engineered with targeting ligands such as antibodies and peptides to improve tumour specificity and uptake. However, excessive loading of ligands can reduce the targeting capabilities of the ligand and reduce the ability of the nanoparticle to bind to a finite number of receptors on cells. Increasing the number of nanoparticles delivered to cells by each targeting molecule would lead to higher signal-to-noise ratios and would improve image contrast. Here, we show that M13 filamentous bacteriophage can be used as a scaffold to display targeting ligands and multiple nanoparticles for magnetic resonance imaging of cancer cells and tumours in mice. Monodisperse iron oxide magnetic nanoparticles assemble along the M13 coat, and its distal end is engineered to display a peptide that targets SPARC glycoprotein, which is overexpressed in various cancers. Compared with nanoparticles that are directly functionalized with targeting peptides, our approach improves contrast because each SPARC-targeting molecule delivers a large number of nanoparticles into the cells. Moreover, the targeting ligand and nanoparticles could be easily exchanged for others, making this platform attractive for in vivo high-throughput screening and molecular detection.

Spatiotemporal controlled delivery of nanoparticles to injured vasculature.

There are a number of challenges associated with designing nanoparticles for medical applications. We define two challenges here: (i) conventional targeting against up-regulated cell surface antigens is limited by heterogeneity in expression, and (ii) previous studies suggest that the optimal size of nanoparticles designed for systemic delivery is approximately 50–150 nm, yet this size range confers a high surface area-to-volume ratio, which results in fast diffusive drug release. Here, we achieve spatial control by biopanning a phage library to discover materials that target abundant vascular antigens exposed in disease. Next, we achieve temporal control by designing 60-nm hybrid nanoparticles with a lipid shell interface surrounding a polymer core, which is loaded with slow-eluting conjugates of paclitaxel for controlled ester hydrolysis and drug release over approximately 12 days. The nanoparticles inhibited human aortic smooth muscle cell proliferation in vitro and showed greater in vivo vascular retention during percutaneous angioplasty over nontargeted controls. This nanoparticle technology may potentially be used toward the treatment of injured vasculature, a clinical problem of primary importance.

M13 Phage-Functionalized Single-Walled Carbon Nanotubes As Nanoprobes for Second Near-Infrared Window Fluorescence Imaging of Targeted Tumors

Second near-infrared (NIR) window light (950-1400 nm) is attractive for in vivo fluorescence imaging due to its deep penetration depth in tissues and low tissue autofluorescence. Here we show genetically engineered multifunctional M13 phage can assemble fluorescent single-walled carbon nanotubes (SWNTs) and ligands for targeted fluorescence imaging of tumors. M13-SWNT probe is detectable in deep tissues even at a low dosage of 2 μg/mL and up to 2.5 cm in tissue-like phantoms. Moreover, targeted probes show specific and up to 4-fold improved uptake in prostate specific membrane antigen positive prostate tumors compared to control nontargeted probes. This M13 phage-based second NIR window fluorescence imaging probe has great potential for specific detection and therapy monitoring of hard-to-detect areas.

Deep, noninvasive imaging and surgical guidance of submillimeter tumors using targeted M13-stabilized single-walled carbon nanotubes.

Significance Early detection of cancer positively impacts diagnosis and treatment, ultimately improving patient survival. Using fluorescence imaging offers the promise of safe, noninvasive detection with excellent resolution and guides surgical removal of tumors to improve patient outcomes. However, the success of current optical probes is limited due to high background from tissue autofluorescence, poor penetration depth, and inherently low signal stability. Here, we engineered M13 bacteriophage to stabilize single-walled carbon nanotubes for selective, targeted imaging of ovarian tumors. These nanoprobes fluoresce at longer near-infrared wavelengths than current probes, thereby improving noninvasive detection of small, deep tumors and guidance for surgical removal of submillimeter tumors. This material-based approach may be attractive to guide surgical interventions where deep tissue molecular imaging is informative. Highly sensitive detection of small, deep tumors for early diagnosis and surgical interventions remains a challenge for conventional imaging modalities. Second-window near-infrared light (NIR2, 950–1,400 nm) is promising for in vivo fluorescence imaging due to deep tissue penetration and low tissue autofluorescence. With their intrinsic fluorescence in the NIR2 regime and lack of photobleaching, single-walled carbon nanotubes (SWNTs) are potentially attractive contrast agents to detect tumors. Here, targeted M13 virus-stabilized SWNTs are used to visualize deep, disseminated tumors in vivo. This targeted nanoprobe, which uses M13 to stably display both tumor-targeting peptides and an SWNT imaging probe, demonstrates excellent tumor-to-background uptake and exhibits higher signal-to-noise performance compared with visible and near-infrared (NIR1) dyes for delineating tumor nodules. Detection and excision of tumors by a gynecological surgeon improved with SWNT image guidance and led to the identification of submillimeter tumors. Collectively, these findings demonstrate the promise of targeted SWNT nanoprobes for noninvasive disease monitoring and guided surgery.

Abstract 4287: M13-stabilized single-walled carbon nanotubes as nanoprobes for second near-infrared window fluorescent imaging of targeted tumors

Second near-infrared (NIR) window light (950-1,400 nm) is attractive for in vivo fluorescence imaging due to its deep penetration depth in tissues and low tissue autofluorescence. Here we show genetically engineered multifunctional M13 phage can assemble fluorescent single-walled carbon nanotubes (SWNTs) and ligands for targeted fluorescence imaging of tumors. M13-SWNT probe is detectable in deep tissues even at a low dosage of 2 μg/mL and up to 2.5 cm in tissue-like phantoms. Moreover, targeted probes show specific and four-fold improved uptake in prostate specific membrane antigen positive prostate tumors compared to control non-targeted probes. This M13 phage-based second NIR window fluorescence imaging probe has great potential for specific detection and therapy monitoring of hard-to-detect areas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4287. doi:1538-7445.AM2012-4287

Abstract 4344: Virus-templated magnetic nanowires for tumor-targeted imaging

In an effort towards early tumor detection, various diagnostic probes, such as tumor marker-specific fluorescent and magnetic nanoparticles, have been developed. Nanoparticles detected using magnetic resonance (MR) imaging is an attractive approach due to its non-invasiveness. Current work has focused on single functionalized spherical nanoparticle systems, one example being cross-linked iron oxide nanoparticles (CLIO). However, these ‘shaped’ particles can be cleared by the reticuloendothelial system upon intravenous injection due to opsonization. Recent work suggests that longer filamentous shaped structures can persist almost 10 times longer in circulation in vivo compared to spherical particles. For clinical diagnostics, it would be desirable to engineer filamentous shaped imaging agents that allow for longer circulation and better homing towards the tumor before immune clearance. M13 filamentous bacteriophage can potentially allow for longer circulation of the imaging agents. Recent work indicates that the virus can circulate in vivo for at least 24 hours compared to the 5-6 hour circulation of various spherical particles (unpublished data). Using the M13 virus as a scaffold, we can uniquely combine multiple nanoparticles with engineered cell-targeting peptide ligands for tumor-targeted imaging. Exploiting the multiple coat proteins on M13 amenable for peptide display, we have genetically engineered peptides that bind iron oxide nanoparticles (γ-Fe2O3 NPs) and that target secreted protein, acidic and rich in cysteine (SPARC), a protein overexpressed in metastatic prostate and breast cancers. We have been able to assemble monocrystalline, monodisperse γ-Fe2O3 NPs along the virus coat and demonstrate enhanced MR contrast properties, compared to commercially available CLIO. We have investigated specific targeting against prostate cancer in vitro and in vivo. There is a decrease in transverse relaxation times (‘dark contrast’) using targeted nanowires compared to negative control and is specific towards SPARC positive C4-2B cell line compared to DU145 control. SPARC-targeted nanowires were injected intravenously in LNCaP and C4-2B and control DU145 xenograft tumors. MR images indicate dark image contrast in SPARC expressing tumors, compared to control. Histology suggests iron accumulation in the stroma and capsule of SPARC-positive tumors compared to control, suggesting tumor targeting in vivo. These virus-templated probes may have potential as long circulating, targeted non-invasive imaging agents for the early detection of tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4344.